Antikine antibodies that bind to multiple cc chemokines

ABSTRACT

An antikine antibody binds to two, three, four, five or more CC chemokines, such as RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, or MCP-1/CCL2. Methods for affinity maturation and humanization of antikine antibodies as well as the production of hybridoma cell lines producing antikine antibodies by sequential immunization are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. application Ser. No.12/870,573, filed Aug. 27, 2010 (allowed), and claims priority under 35U.S.C. 119(e) to U.S. Provisional Application 61/238,015, filed Aug. 28,2009. Each of these applications is hereby incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention encompasses antikine antibodies, or antibodies that bindto two, three, four, five, or more CC chemokines (CC chemokines are alsoknown as β-chemokines), particularly those antibodies which bind to atleast two chemokines selected from the group consisting of RANTES/CCL5,MIP-1α/CCL3, MIP-1β/CCL4, and MCP-1/CCL2. Unlike antibodies that bind toonly a single CC chemokine, antikine antibodies practically address theproblem of functional redundancy amongst CC chemokines by binding to,detecting, and/or neutralizing more than one CC chemokine at a time.Other aspects of the invention include diagnostic and therapeutic usesof antikine antibodies including the treatment of conditions, disorders,or diseases mediated by CC chemokines; hybridoma cell lines producingantikine antibodies and methods for producing hybridomas by sequentialimmunization; methods for humanizing antikine antibodies; and methodsfor improving antikine antibodies by affinity maturation.

2. Description of the Related Art

Chemokines are key mediators of inflammation and are implicated in thedevelopment of autoimmune disease; Viola & Luster, Ann. Rev. Pharmacol.Toxicol. 48: 171-197 (2008). They are produced at sites of inflammationor infection and induce the migration of leukocytes from the circulationinto the tissue. Simple and effective ways for modulating inflammationor immunological processes mediated by chemokines have long been sought.These efforts are complicated by the redundancy and overlap in thefunctions of numerous different chemokines and their receptors. Forexample, chemokine inhibitors have been used to treat autoimmuneconditions in preclinical animal models, but have yet to succeed in theclinic for treating autoimmune indications. It has been proposed thatthis lack of efficacy may be due to the redundancy in the functions ofchemokines. Over 50 different chemokines have been identified and eachhas different structural and functional properties. The specificity ofsome chemokines overlap, that is, they bind to the same type of receptoror act on similar types of cells; Vergunst, et al., Arthritis Rheum. 58:1931-1939 (2008). A particular chemokine may bind to more than one typeof chemokine receptor and a given chemokine receptor may be bound bymore than one type of chemokine. Therefore, the development of a singleagent capable of binding to, blocking chemokine binding to a receptor,or otherwise neutralizing the activity of more than one chemokine ishighly desirable.

Chemokines which take their name from chemotactic cytokines, are smallsecreted polypeptides that regulate movement of immune cells intotissues; Baggiolini, et al., Adv. Immunol. 55:97-179 (1994); Oppenheimet al., Ann. Rev. Immunol. 9:617-648 (1991).

All chemokines share a Greek key structure that is stabilized bydisulfide bonds between conserved cysteine residues. However, chemokinesare further assigned to four different families based on the number andposition of these conserved cysteine residues. The α- and β-chemokineseach contain four conserved cysteine residues. The first two cysteinesof an α-chemokine are separated by a single amino acid, thus forming acharacteristic CXC amino acid motif. The first two conserved cysteinesof a β-chemokine are adjacent. Thus, the β-chemokines are also known asCC chemokines. By contrast, lymphotactin is the sole member of a thirdclass of XC chemokines and contains only the second and fourth conservedcysteine residues. A fourth class of chemokines, of which fractalkine isthe sole member, is the CXXXC or CX₃C class which has 3 amino acidsseparating the first two conserved cysteines. In humans, α-chemokinesare mainly encoded by genes clustered on chromosome 4 and β-chemokinesare mainly encoded by genes on chromosome 17. Lymphotactin is encoded onchromosome 1 and fractalkine on chromosome 16.

Chemokines form gradients that serve as chemoattractants and potentialproliferation signals for immune and other cells such as monocytes,macrophages, basophils, eosinophils, T lymphocytes and fibroblasts. CCchemokines exhibit chemoattractant properties by forming concentrationgradients recognized by chemotactic cells; CC chemokines also signalparticular cell types to proliferate, including fibroblasts and immunecells such as monocytes, macrophages, T lymphocytes, basophils, andeosinophils. The target receptors and cells of chemokines, including CCchemokines are described by Viola, et al., Annu Rev. Pharmacol. Toxicol.48:171-197 (2008), see e.g., FIG. 1, which document is herebyincorporated by reference for its teachings regarding CC chemokinechemoattractant and signaling functions.

Chemokines share structural features associated with particularchemokine functions, such as with binding to a chemokine receptor.Common structures include an elongated N-terminus segment (N terminaldomain) that precedes the first cysteine residue, N loop, 3₁₀ helix,beta strands β1, β2, and β3, the 30's, 40's and 50s loops; location ofdisulfide bonds, and the C-terminal α-helical segment.

These and other CC chemokine structures, including conserved orhomologous amino acid residues among different CC chemokines, as well asthe solvent-accessible, partially solvent accessible, and buried aminoacid residues of CC chemokines are incorporated by reference Fernandez,et al., Annu. Rev. Pharmacol. Toxicol. 42:469-99 (2002), see e.g., FIGS.1 and 2. Buried amino acid residues of intact, undenatured chemokinesare unlikely to form epitopes or antigenic determinants contacted byantibodies to a chemokine. In contrast, solvent or surface-exposed CCchemokine residues are more accessible to antibody binding.

CC chemokine residues associated with chemokine receptor binding ofCCL3/MIP-1α include residues 11-15 (CCFSY), residues 17-24 (SRQIPQNF),residues 34-35 (QC), and residues 57-67 (EWVQKYVSDLE) of SEQ ID NO: 71;

residues associated with CCL4/MIP-1β chemokine receptor binding includeresidues 11-15 (CCFSY), residues 17-24 (ARKLPHNF), residues 34-35 (LC),or residues 57-67 (SWVQEYVYDLE) of SEQ ID NO: 72;

residues associated with CCL5/RANTES binding include residues 10-14(CCFAY), residues 16-23 (ARPLPRAH), residues 33-34 (KC), or residues56-66 (KWVREYINSLE) of SEQ ID NO: 73;

residues associated with CCL23/MPIF-1 binding include residues 9-13(CCISY), residues 15-22 (PRSIPCSL), residues 32-33 (EC), or residues55-65 (KQVQVCMRMLK) of SEQ ID NO: 81; and

residues associated with CCL15/HCC-2 include residues 8-12 (CCTSY),residues 14-21 (SQSIPCSL), residues 31-32 (EC), or residues 54-64(PGVQDCMKKLK) of SEQ ID NO: 79. Corresponding amino acid residues ofother CC chemokines are depicted, for example, by FIG. 1 of Fernandez,et al., id. (2002).

Conserved domains for CC chemokines are disclosed athttp://www.ncbi.nlm.nih.gov/. This structural data is incorporated byreference to the protein and conserved domain database information atthe website named above as last accessed Aug. 24, 2010.

Chemokines in the CC chemokine class interact with seven transmembraneG-protein coupled receptors termed CC chemokine receptors or CCRs, Rossi& Zlotnik, Ann. Rev. Immunol. 18:217-242 (2002). Interaction of thechemokine with its receptor regulates activation of adhesion moleculesand affects diapedesis and extravasation of immune cells from thecirculation into tissues.

Chemokines have been implicated in the development and maintenance ofnumerous inflammatory and immunological conditions, disorders anddiseases. These include rheumatoid arthritis, multiple sclerosis,atherosclerosis, psoriasis, inflammatory bowel disease (includingCrohn's disease, ulcerative colitis, Celiac disease), vascularrestenosis, lupus nephritis, glomerulonephritis, transplant rejection,scleroderma, fibrotic disease, asthma (and other lung inflammatoryconditions). For example, levels of CC-chemokines are elevated inaffected tissues from patients with rheumatoid arthritis, multiplesclerosis (MS), atherosclerosis, and others. Preclinical animal modelsof these diseases show that inhibition of individual chemokines can atleast partially ameliorate disease symptoms. For example, Kasama, etal., J. Clin. Invest. 95: 2868-2876 (1995) demonstrated thatadministration of an antibody which inhibited MIP-1α/CCL3 could reducearthritis clinical scores by approximately 50% in a rodent model ofrheumatoid arthritis. Similarly, Ogata, et al., J. Pathol. 182: 106-114(1997) showed that an anti-MCP-1/CCL2 antibody could decrease jointswelling by approximately 30%. Receptors for MIP-1α/CCL3 (including CCR1and CCR5) and MCP-1/CCL2 (CCR2) are expressed in an overlapping patternin leukocytes, and thus it is possible that an inhibitor of bothMIP-1α/CCL3 and MCP-1/CCL2 could be more efficacious than individualinhibitors of each chemokine alone. Viola, et al., Annu. Rev. Pharmacol.Toxicol. 48:171-197 (2008), is hereby incorporated by reference for itsdisclosure of specific classes or types of diseases and disordersassociated with or mediated by such CC chemokines, see e.g., Table 1.

Natural inhibitors of chemokine activity are known and specific agents,such as antibodies or small molecule inhibitors that bind to orinterfere with the activity of particular chemokines have beendeveloped, see Fernandez, et al., Annu. Rev. Pharmacol. Toxicol. 42:469-99 (2002), to which such inhibitors and agents are incorporated byreference, see e.g., pages 482-488. Vaccinia and related pox virusesproduce a soluble 35 kD protein termed vCCI (SEQ ID NO: 117) which bindsand inhibits multiple chemokines within the CC class of chemokines. TheCC class of chemokines generally acts upon leukocytes including T cellsand monocytes; Smith et al., Virology 236: 316-327 (1997), Burns et al.,J. Biol. Chem. 277: 2785-2789 (2002). Recombinant vCCI has been shown tobe effective in reducing leukocyte infiltration in several models ofchronic inflammatory disease, including experimental autoimmuneencephalitis; Jones et al., Cytokine 43: 220-228 (2008) and asthma;Dabbagh, et al., J. Immunol. 165: 3418-3422 (2000). However, use ofnatural substances such as viral proteins like vCCI foreign to asubject's immune system raises safety issues. Administration ofsubstances, such as vCCI can induce undesired physiological or immuneresponses and such substances can be neutralized, removed or destroyedas foreign by host clearance or defense mechanisms.

With this in mind, the inventors focused on developing a method forproducing antibodies, especially humanized antibodies, that canspecifically bind to and neutralize more than one chemokine, but whichdo not pose the risks associated with molecules like vCCI. Prior artchemokine inhibitors, such as antibodies that bind to a single CCchemokine suffer from the problem of CC chemokine receptor redundancy.For example, CCL3/MIP-1α and CCL5/RANTES each bind to chemokinereceptors 1 (CCR1) and 5 (CCR5), see FIG. 1 of Viola, id. (2008). Anantibody that only inhibits CCL3 binding to these chemokine receptorswouldn't prevent receptor activation by the binding of other CCchemokines, such as CCL5.

The inventors initially targeted CC chemokines MIP-1α/CCL3, MIP-1β/CCL4and RANTES/CCL5, which constitute the primary ligands for chemokinereceptors CCR1 and CCR5. CCL2/MCP-1 was also targeted as a ligand forCCR2. As shown by FIG. 1 of Viola, et al., id. (2008), these receptorsare broadly expressed on monocytes and T cells, as well as on otherleukocyte subsets. They are implicated in numerous inflammatory diseasestates both in preclinical animal models of disease and in humandisease.

BRIEF DESCRIPTION OF THE INVENTION

One aspect of the invention is an isolated antikine antibody or antigenbinding fragment thereof that can bind to at least two, three, four,five, six or seven or more different CC chemokines. One example of anantikine antibody is one that can bind to two or more CC chemokines,including at least one CC chemokine that interacts with chemokinereceptor CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10.In another embodiment, an antikine will bind to at least two or three ofCCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, and CCL2/MCP-1 which interactwith CCR1, CCR2 and CCR5. Other examples of an antikine antibody orantigen binding fragment of an antikine antibody include those bindingto at least three different CC chemokines selected from the groupconsisting of CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES,CCL14/HCC-1, CCL15/HCC-2, CCL18/PARC, and CCL23/MPIF-1.

Binding occurs via contact between the antikine antibody and at leastone determinant of the CC chemokine. For example, binding may occurbetween the antibody and a determinant of CCL2/MCP-1, CCL3/MIP-1α,CCL4/MIP-1β, CCL5/RANTES, CCL14/HCC-1, CCL15/HCC-2, CCL18/PARC, orCCL23/MPIF-1 that is located between the CC residues of the CC-chemokineand the last C residue of the CC chemokine. The location of the adjacentCC (cysteine-cysteine) residues and the last cysteine residue in CCchemokines is well known in the art and may be easily identified in thesequences of CC chemokines shown in the sequence listing.

Antikine antibodies and their antigen binding fragments can also bind toat least one determinant of a CC chemokine, including CCL2/MCP-1,CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL14/HCC-1, CCL15/HCC-2,CCL18/PARC, and CCL23/MPIF-1, which is located in the N-loop, 30's loop,or 40's loop of said CC chemokine.

An antikine antibody may also be characterized by its ability to bind tosome CC chemokines but not to others. For example, an antikine antibodymay bind to CCL3/MIP-1α and CCL4/MIP-1β, but not bind to CCL5/RANTES,CCL2/MCP-1, CCL8/MCP-2 or CCL7/MCP-3. Others will not bind to some orall of the chemokines and other biologically active molecules named inthe figures including MIP-1α, MIP-1β, RANTES, MPIF-1, HCC-1, HCC-2,HCC-4, Parc, MCP-2, MCP-3, MCP-4, Eotaxin, MDC, ELC, 1-309, IL-8, SDF orFractalkine, as well as to other chemokines. For example, antikineswhich do not bind to at least one of MCP-1, MCP-2 or MCP-3 areexemplified herein.

An antikine may also be characterized by an ability to bind to two ormore CC chemokine of one species, but not to a corresponding CCchemokine of another species. For example, an antikine may bind to humanCCL3, but not substantially bind to murine CCL3. Specific examples ofbinding specificity of antikine antibodies appear in FIGS. 8-12.

Some antikine antibodies and their antigen-binding fragments can alsoinhibit the interaction of a CC chemokine with a corresponding receptor.Such inhibition may have functional effects, such as inhibitingchemotaxis or other effects activated by chemokine binding to areceptor.

The antikine antibody may bind to at least one determinant within CCreceptor binding residues of a CC chemokine. Receptor binding amino acidresidues and segments of CC chemokines thought or known to be associatedwith CC chemokine receptor binding are documented in the art.

The N-terminus, N-loop, 30s-loop, and residues next to the disulfidesand in the alpha helix are thought to be involved in receptor bindingfor the CC chemokine family in general. Description of particularstructural features of chemokines that correlate to their variousfunctions is incorporated by reference to the following twopublications. Baysal, et al., Proteins 43(2):150-60 (2001) and Kuloglu,et al., Biochemistry, 40(42):12486-96 (2001). See NCBI Conserved DomainDatabase CDD 29111 for the proposed functional domains of CC chemokines.

The inventors have produced and identified specific antikine monoclonalantibodies 3C12F, 7D1G, 7D12A, 18V4F and 18P7E. These antikines may beused to identify other antibodies or substances that competitively blockthe binding of these antikine monoclonal antibodies to one or more ofthe CC chemokines they recognized using competitive inhibition assaysknown in the art. Competitive inhibitors, such as antibodies thatinhibit antikine antibody binding are also encompassed by the inventionas well as methods of detecting such competitive inhibitors usingantikine monoclonal antibodies 3C12F, 7D1G, 7D12A, 18V4F and 18P7E.

Antikine antibodies may be human antibodies, humanized antibodies,chimeric human-murine antibodies, murine or other vertebrate, avian ormammalian antibodies, or their antigen binding fragments.

One type of antikine antibody of the invention has binding specificitiesidentical or similar to that of monoclonal antibody 3C12F and can bindto at least two, three, four or five CC chemokines selected from thegroup consisting of CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL15/HCC-2,and CCL23/MPIF-1. These may exhibit no or substantially no binding toother chemokines, including other chemokines shown in FIG. 8, such asHCC-1, PARC, or MCP 1, 2 or 3. Antibodies of this type may bind todomains which may be important in binding to chemokine receptors,including:

a determinant in the N loop, 30s loop, or 40's loop of a CC chemokine;

at least one antigenic determinant of CCL3/MIP-1α located withinresidues 11-15 (CCFSY), residues 17-24 (SRQIPQNF), residues 34-35 (QC),or residues 57-67 (EWVQKYVSDLE) of SEQ ID NO: 71;

at least one antigenic determinant of CCL4/MIP-1β located withinresidues 11-15 (CCFSY), residues 17-24 (ARKLPHNF), residues 34-35 (LC),or residues 57-67 (SWVQEYVYDLE) of SEQ ID NO: 72;

at least one antigenic determinant of CCL5/RANTES located withinresidues 10-14 (CCFAY), residues 16-23 (ARPLPRAH), residues 33-34 (KC),or residues 56-66 (KWVREYINSLE) of SEQ ID NO: 73;

at least one antigenic determinant of CCL23/MPIF-1 located withinresidues 9-13 (CCISY), residues 15-22 (PRSIPCSL), residues 32-33 (EC),or residues 55-65 (KQVQVCMRMLK) of SEQ ID NO: 81; or

at least one antigenic determinant of CCL15/HCC-2 located withinresidues 8-12 (CCTSY), residues 14-21 (SQSIPCSL), residues 31-32 (EC),or residues 54-64 (PGVQDCMKKLK) of SEQ ID NO: 79.

This type of antibody may comprise at least one complementaritydetermining region (CDR) of MAb 3C12F selected from the group consistingof SEQ ID NOS: 3, 4, 5, 8, 9 or 10, or SEQ ID NOS: 53, 54, 55, 58, 59 or60 or at least one CDR of an antibody that competitively inhibits orblocks the binding of MAb 3C12F to the CC chemokines it binds or blocksbinding of RANTES to vCCI (see FIG. 2). An antikine antibody of thistype may contain 1, 2, 3, 4, 5 or 6 CDRs of MAb 3C12F or CDRs in which1, 2, 3, 4, 5, 6, 7, 8 or more amino acid residues of SEQ ID NOS: 3, 4,5, 8, 9 and/or 10, or SEQ ID NOS: 53, 54, 55, 58, 59 and/or 60 have beendeleted, inserted or substituted. Thus, CDR sequences may be identicalto those of an antibody produced by hybridoma cell line 3C12F or by asubculture thereof; may correspond to those of an antikine antibodyanalog of 3C12F, or correspond to those of an antikine monoclonalantibody that competitively blocks or inhibits the binding of MAb 3C12Fto two or more of the CC chemokines to which it binds. Such antibodiesmay be in the form of a human antibody, a humanized antibody, a chimerichuman-murine antibody, murine, avian or other vertebrate antibody; or anantigen binding fragment thereof.

A second type of antibody has a binding specificity that is similar oridentical to 7D1G, and binds to at least two, three, four, five or sixCC chemokines selected from the group consisting CCL3/MIP-1α,CCL4/MIP-1β, CCL5/RANTES, CCL14/HCC-1, CCL23/MPIF-1, and CCL18/PARC.This type of antibody may exhibit no or substantially no binding toother chemokines, such as the other chemokines shown in FIG. 10, such asFICC-2, Eotaxin, or MCP 1, 2, 3 or 4.

Antibodies of this second type may bind to structural determinants of achemokine or CC chemokine,

including to a determinant in the N loop or 40's loop of at least one ofthe CC chemokines bound by MAb 7D1G;

at least one antigenic determinant of CCL3/MIP-1α located withinresidues 11-15 (CCFSY), residues 17-24 (SRQIPQNF), residues 34-35 (QC),or residues 57-67 (EWVQKYVSDLE) of SEQ ID NO: 71;

at least one antigenic determinant of CCL4/MIP-1β located withinresidues 11-15 (CCFSY), residues 17-24 (ARKLPHNF), residues 34-35 (LC),or residues 57-67 (SWVQEYVYDLE) of SEQ ID NO: 72;

at least one antigenic determinant of CCL5/RANTES located withinresidues 10-14 (CCFAY), residues 16-23 (ARPLPRAH), residues 33-34 (KC),or residues 56-66 (KWVREYINSLE) of SEQ ID NO: 73;

at least one antigenic determinant of CCL23/MPIF-1 located withinresidues 9-13 (CCISY), residues 15-22 (PRSIPCSL), residues 32-33 (EC),or residues 55-65 (KQVQVCMRMLK) of SEQ ID NO: 81;

at least one antigenic determinant of CCL14/HCC-1 residues 8-12 (CCFTY),residues 14-21 (TYKIPRQR), residues 31-32 (QC), or residues 54-64(KWVQDYIKDMK) of SEQ ID NO: 78; or

at least one antigenic determinant of CCL18/PARC residues 10-14 (CCLVY),residues 16-23 (SWQIPQKF), residues 33-34 (QC), or residues 56-66(KWVQKYISDLK) of SEQ ID NO: 82.

Structurally, these antibodies may comprise one or more CDRs of MAb7D1G, selected from the group consisting of SEQ ID NOS: 23, 24, 25, 28,29 or 30, or a CDR from an antibody that competitively inhibits orblocks the binding of MAb 7D1G to the CC chemokines it binds. One kindof an antikine antibody of this type will contain 1, 2, 3, 4, 5 or 6CDRs of MAb 7D1G or CDRs in which 1, 2, 3, 4, 5, 6, 7, 8 or more aminoacid residues of SEQ ID NOS: 23, 24, 25, 28, 29 and/or 30 have beendeleted, inserted or substituted. Thus, CDR sequences may be identicalto those of an antibody produced by hybridoma cell line 7D1G or by asubculture thereof; may correspond to those of an antikine antibodyanalog of 7D1G, or correspond to those of an antikine monoclonalantibody that competitively blocks or inhibits the binding of MAb 7D1Gto two or more of the CC chemokines to which it binds. Such antibodiesmay be in the form of a human antibody, a humanized antibody, a chimerichuman-murine antibody, murine, avian or other vertebrate antibody; or anantigen binding fragment thereof.

A third type of antikine antibody has a binding specificity similar oridentical to 7D12A and binds to at least two, three or four CCchemokines selected from the group consisting of CCL3/MIP-1α,CCL4/MIP-1β, CCL5/RANTES, and CCL23/MPIF-1. These may exhibit no orsubstantially no binding to other chemokines, including other chemokinesshown in FIG. 9, such as CCL2/MCP-1.

Such an antibody product may bind to a determinant in the N loop or 40'sloop of at least one of said CC chemokines; may bind to at least oneantigenic determinant of CCL3/MIP-1α located within residues 11-15(CCFSY), residues 17-24 (SRQIPQNF), residues 34-35 (QC), or residues57-67 (EWVQKYVSDLE) of SEQ ID NO: 71;

at least one antigenic determinant of CCL4/MIP-1β located withinresidues 11-15 (CCFSY), residues 17-24 (ARKLPHNF), residues 34-35 (LC),or residues 57-67 (SWVQEYVYDLE) of SEQ ID NO: 72;

at least one antigenic determinant of CCL5/RANTES located withinresidues 10-14 (CCFAY), residues 16-23 (ARPLPRAH), residues 33-34 (KC),or residues 56-66 (KWVREYINSLE) of SEQ ID NO: 73; or

at least one antigenic determinant of CCL23/MPIF-1 residues 9-13(CCISY), residues 15-22 (PRSIPCSL), residues 32-33 (EC), or residues55-65 (KQVQVCMRMLK) of SEQ ID NO: 81.

Structurally these third type antibodies may comprise at least one CDRof MAb 7D12A selected from the group consisting of SEQ ID NOS: 13, 14,15, 18, 19 or 20, or at least one CDR from an antibody thatcompetitively inhibits the binding of MAb 7D12A to CC chemokines itbinds. An antikine antibody of this type may contain 1, 2, 3, 4, 5 or 6CDRs of MAb 7D12A or CDRs in which 1, 2, 3, 4, 5, 6, 7, 8 or more aminoacid residues of SEQ ID NOS: 13, 14, 15, 18, 19 and/or 20, have beendeleted, inserted or substituted. Thus, CDR sequences may be identicalto those of an antibody produced by hybridoma cell line 7D12A or by asubculture thereof; may correspond to those of an antikine antibodyanalog of 7D12A, or correspond to those of an antikine monoclonalantibody that competitively blocks or inhibits the binding of MAb 7D12Ato two or more of the CC chemokines to which it binds. Such antibodiesmay be in the form of a human antibody, a humanized antibody, a chimerichuman-murine antibody, murine, avian or other vertebrate antibody; or anantigen binding fragment thereof.

A fourth type of antikine antibody has a binding specificity similar oridentical to 18V4F and binds to at least two or three CC chemokinesselected from the group consisting of CCL3/MIP-1α, CCL4/MIP-1β, andCCL5/RANTES. These may exhibit no or substantially no binding to otherchemokines, including other chemokines shown in FIG. 11, such asCCL2/MCP-1. Such an antibody product may bind to a determinant in the Nloop or 40's loop of at least one of said CC chemokines; may bind to atleast one antigenic determinant of CCL3/MIP-1α located within residues11-15 (CCFSY), residues 17-24 (SRQIPQNF), residues 34-35 (QC), orresidues 57-67 (EWVQKYVSDLE) of SEQ ID NO: 71;

at least one antigenic determinant of CCL4/MIP-1β located withinresidues 11-15 (CCFSY), residues 17-24 (ARKLPHNF), residues 34-35 (LC),or residues 57-67 (SWVQEYVYDLE) of SEQ ID NO: 72;

at least one antigenic determinant of CCL5/RANTES located withinresidues 10-14 (CCFAY), residues 16-23 (ARPLPRAH), residues 33-34 (KC),or residues 56-66 (KWVREYINSLE) of SEQ ID NO: 73.

Structurally this fourth type of antibody may comprise at least one CDRof MAb 18V4F selected from the group consisting of SEQ ID NOS: 33, 34,35, 38, 39, or 40 or SEQ ID NOS: 63, 64, 65, 68, 69 or 70, or at leastone CDR from an antibody that competitively inhibits the binding of MAb18V4F to CC chemokines it binds. An antikine antibody of this type maycontain 1, 2, 3, 4, 5 or 6 CDRs of MAb 18V4F or CDRs in which 1, 2, 3,4, 5, 6, 7, 8, or more amino acid residues of 33, 34, 35, 38, 39 and/or40; or SEQ ID NOS: 63, 64, 65, 68, 69 and/or 70 have been deleted,inserted or substituted with other amino acid residues. Thus, CDRsequences may be identical to those of an antibody produced by hybridomacell line 18V4F or by a subculture thereof; may correspond to those ofan antikine antibody analog of 18V4F, or correspond to those of anantikine monoclonal antibody that competitively blocks or inhibits thebinding of MAb 18V4F to two or more of the CC chemokines to which itbinds. Such antibodies may be in the form of a human antibody, ahumanized antibody, a chimeric human-murine antibody, murine, avian orother vertebrate antibody; or an antigen binding fragment thereof.

A fifth type of antikine antibody has a binding specificity similar oridentical to 18P7E and binds to at least two, or three CC chemokinesselected from the group consisting of CCL3/MIP-1α, CCL4/MIP-1β, andCCL5/RANTES. These may exhibit no or substantially no binding to otherchemokines, including other chemokines shown in FIG. 12, such asCCL2/MCP-1. Such an antibody product may bind to a determinant in the Nloop or 40's loop of at least one of the three CC chemokines mentionedabove.

Such an antibody product may bind to a determinant in the N loop or 40'sloop of at least one of said CC chemokines; may bind to

at least one antigenic determinant of CCL3/MIP-1α located withinresidues 11-15 (CCFSY), residues 17-24 (SRQIPQNF), residues 34-35 (QC),or residues 57-67 (EWVQKYVSDLE) of SEQ ID NO: 71;

at least one antigenic determinant of CCL4/MIP-1β located withinresidues 11-15 (CCFSY), residues 17-24 (ARKLPHNF), residues 34-35 (LC),or residues 57-67 (SWVQEYVYDLE) of SEQ ID NO: 72;

at least one antigenic determinant of CCL5/RANTES located withinresidues 10-14 (CCFAY), residues 16-23 (ARPLPRAH), residues 33-34 (KC),or residues 56-66 (KWVREYINSLE) of SEQ ID NO: 73.

Structurally this fifth type of antibody may comprise at least one CDRof MAb 18P7E selected from the group consisting of SEQ ID NOS: 43, 44,45, 48, 49 or 50, or at least one CDR from an antibody thatcompetitively inhibits the binding of MAb 18P7E to CC chemokines itbinds. An antikine antibody of this type may contain 1, 2, 3, 4, 5 or 6CDRs of MAb 18P7E or CDRs in which 1, 2, 3, 4, 5, 6, 7, 8 or more aminoacid residues of ID NOS: 43, 44, 45, 48, 49 and/or 50 have been deleted,inserted or substituted with other amino acids. Thus, CDR sequences maybe identical to those of an antibody produced by hybridoma cell line18P7E or by a subculture thereof; may correspond to those of an antikineantibody analog of 18P7E, or correspond to those of an antikinemonoclonal antibody that competitively blocks or inhibits the binding ofMAb 18P7E to two, three or more of the CC chemokines to which it binds.Such antibodies may be in the form of a human antibody, a humanizedantibody, a chimeric human-murine antibody, murine, avian or othervertebrate antibody; or an antigen binding fragment thereof.

The light and heavy variable domain sequences, including those of eachCDR, of 3C12F, 7D1G, 7D12A, 18V4F, 18P7E and their analogs, or otherantikine antibodies, may be employed as a core structure for drug designof antibody mimics, as competitive inhibitors which interfere with CCchemokine-receptor binding, as ligands to isolate or identify chemokinesor anti-ithotypic antibodies to CC chemokine antibodies, or asimmunogens to induce anti-idiotype antibodies against antibodies to CCchemokines. Such peptides include modified or stabilized peptides orconformationally constrained peptides, such as a circular or loopedpeptide comprising the CDRs of an antikine antibody. Methods of peptidedesign using the CDR of antibodies are known in the art and areincorporated by reference to Takahashi, et al., Chem. Eur. J.6(17):3196-3203 or Feng, et al., Cell. Host. Microb. 98(2): 311-316.These CDRs include those of SEQ ID NOS: 3-5, 8-10, 13-15, 18-20, 23-25,28-30, 33-35, 38-40, 43-45, 48-50, 53-55, 58-60, 63-65 and 68-70 as wellas analogs of these peptide sequences produced by affinity maturation.Combinations of different CDRs either as combinations of separatepeptides comprising different CDRs, or as a conjugate, hybrid, or fusionof two or more CDR peptide sequences to form a unitary peptide productmay be used to modulate or inhibit CC chemokine binding or activity,inhibit chemokine dimer- or multimerization, or induce usefulphysiological or immunological responses.

The antikine antibodies of the invention may be formulated as acomposition comprising the antikine antibody or its antigen bindingfragments with a carrier, excipient or buffer as described in moredetail below.

Methods for making hybridoma cell lines producing antikine antibodiesinclude sequentially immunizing a mammal, such as a mouse, with aparticular CC chemokine, followed by boosting with one or more differentCC chemokines, and then producing a hybridoma cell line from saidmammal, for example by fusion of its spleen cells with a myeloma orimmortalized B cell line, and isolating a hybridoma cell line thatproduces an antikine antibody binding to two or more chemokines or CCchemokines.

Methods for treating a disease, disorder or condition mediated by one ormore CC chemokines, especially those mediated by at least two or threeCC chemokines recognized by an antikine antibody comprise administeringto a subject in need thereof the antikine antibody or an antigen bindingfragment thereof. The disease, disorder, or condition may becharacterized by inflammation or by autoimmunity.

Other aspects of the invention will be apparent from the drawings andthe detailed description of the embodiments which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts binding of purified 3C12F to chemokines by ELISA.

FIG. 2 shows that 3C12F blocks vCCI binding to RANTES/CCL5.

FIG. 3 shows titration of inhibitory activity of purified 3C12F inchemotaxis assays

FIG. 4 shows titration of inhibitory activity of purified 7D12A inchemotaxis assays.

FIG. 5 shows titration of inhibitory activity of purified 7D1G inchemotaxis assays.

FIG. 6 shows titration of inhibitory activity of purified 18V4F inchemotaxis assays.

FIG. 7 shows titration of inhibitory activity of purified 18P7E inchemotaxis assays.

FIG. 8 depicts the chemokine binding specificity of 3C12F using the MSDplatform.

FIG. 9 depicts the chemokine binding specificity of 7D12A using the MSDplatform.

FIG. 10 depicts the chemokine binding specificity of 7D1G using the MSDplatform.

FIGS. 11A and 11B depict the chemokine binding specificity of 18V4Fusing the MSD platform.

FIG. 12 depicts the chemokine binding specificity of 18P7E using the MSDplatform.

FIGS. 13A, 13B, 13C, and 13D show alignments of the chemokine sequencesrecognized by the five antikine antibodies.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibody” is to be construed broadly as describing singlemonoclonal antibodies, antibody compositions with polyepitopicspecificity, as well as antibody fragments (e.g., Fab, F(ab′)₂, scFv andFv), as long as they exhibit the desired biological activity, such as anability to bind to a particular antigen, epitope, or antigenicdeterminant. This term includes intact antibodies, full-length ornon-truncated antibodies, as well as antibody fragments, and antibodyderivatives, variants and analogs.

Antibodies, including antikine antibodies described below, may havedifferent isotypes—e.g., IgA, IgD, IgE, IgG, IgM, and IgY—as well asvarious isotype subclasses, such as human IgG subclasses 1, 2, 3 and 4or IgA subclasses 1 and 2. Multivalent antibodies may be characterizedby their avidity for a multivalent antigen. Avidity strengthens bindingto antigens with repeating identical epitopes, and some chemokines havebeen characterized by dimeric or tetrameric structures. The moreantigen-binding sites an individual antibody molecule has, the higherits avidity for antigen. Antibodies may be obtained or derived fromvarious vertebrates, including those of mammals and birds.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Köhler & Milstein, Nature, 256:495 (1975), or may be madeby recombinant DNA methods, see e.g., Cabilly, et al., U.S. Pat. No.4,816,567.

A “chimeric antibody” refers to antibodies containing amino acidsequences from two different sources, e.g., one that contains conservedhuman antibody segments spliced to variable segments of a murineantibody known to bind to a particular epitope or antigen. One portionof each of the amino acid sequences of heavy and light chains isidentical or homologous to sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chains is homologous or identical to sequencesfrom another species. In one embodiment, the invention features achimeric antibody or antigen-binding fragment, in which the variableregions of both light and heavy chains mimic the variable regions ofantibodies derived from one species of mammals, while the constantportions are homologous to the sequences in antibodies derived fromanother species. In one embodiment of the invention, chimeric antibodiesare made by grafting CDRs from a mouse antibody onto the frameworkregions of a human antibody. Thus, the monoclonal antibodies of theinvention include “chimeric” antibodies in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies retaining anability to bind to a CC chemokine. The features of chimeric antibodiesand methods for making them are incorporated by reference to Cabilly, etal., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.Sci. USA, 81:6851-6855 (1984).

A “complementarity determining region (CDR)” forms part of a variableregion of a light or heavy chain of an immunoglobulin molecule. Thesesections of an antibody chain form a portion of the region whichdetermines specificity of an antibody for a particular epitope on anantigen and form portions of the antibody molecule that may directlybind to the epitope. CDR3 shows the most variability amongst thedifferent CDRs forming an antibody. CDRs mediate contact between anantibody and the epitope it recognizes. Isolated peptides comprising oranalogous to CDR sequences may exhibit additional functional activities:Polonelli, et al., PLoS One 3:e2371 (2008).

“Humanized antibodies” refer to antibodies which comprise at least onechain comprising variable region framework residues substantially from ahuman antibody chain (referred to as the acceptor immunoglobulin orantibody) and at least one complementarity determining region (CDR)substantially from a non-human-antibody (e.g., mouse). In addition tothe grafting of the CDRs, humanized antibodies typically undergo furtheralterations in order to improve affinity and/or decrease immunogenicity.A “humanized antibody” encompasses not completely human antibodies thatare chimeric antibodies which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornon-human primate having the desired specificity, affinity, andcapacity. In some instances, Framework Region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin thatimmunospecifically binds to two or more CC chemokines that has beenaltered by the introduction of amino acid residue substitutions,deletions or additions (i.e., mutations). In some embodiments, ahumanized antibody is a derivative. Such a humanized antibody comprisesamino acid residue substitutions, deletions or additions in one or morenon-human CDRs. The humanized antibody derivative may have substantiallythe same binding, better binding, or worse binding when compared to anon-derivative humanized antibody. In specific embodiments, one, two,three, four, or five amino acid residues of the CDR have beensubstituted, deleted or added (i.e., mutated). Methods for producinghumanized antibodies are incorporated by reference to European PatentNos. EP 239,400, EP 592,106, and EP 519,596; International PublicationNos. WO 91/09967 and WO 93/17105; U.S. Pat. Nos. 5,225,539, 5,530,101,5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, MolecularImmunology 28(4/5):489-498 (1991); Studnicka, et al., ProteinEngineering 7(6):805-814 (1994); Roguska, et al., Proc. Natl. Acad. Sci.USA 91:969-973 (1994); Tan, et al., J. Immunol. 169:1119-25 (2002);Caldas, et al., Protein Eng. 13:353-60 (2000); Morea, et al., Methods20:267-79 (2000); Baca, et al., J. Biol. Chem. 272:10678-84 (1997);Roguska, et al., Protein Eng. 9:895-904 (1996); Couto, et al., CancerRes. 55 (23 Supp):5973s-5977s (1995); Couto, et al., Cancer Res.55:1717-22 (1995); Sandhu, Gene 150:409-10 (1994); Pedersen, et al., J.Mol. Biol. 235:959-73 (1994); Jones, et al., Nature 321:522-525 (1986);Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992).

A “variant” or “analog” antibody, refers herein to a molecule whichdiffers in amino acid sequence from a “parent” antibody amino acidsequence by virtue of addition, deletion and/or substitution of one ormore amino acid residue(s) in the parent antibody sequence. In oneembodiment, the variant comprises one or more amino acid substitution(s)in one or more hypervariable region(s) of the parent antibody. Forexample, the variant may comprise at least one, e.g. from about one toabout ten, and preferably from about two to about five, substitutions inone or more hypervariable regions of the parent antibody. Ordinarily,the variant will have an amino acid sequence having at least 75% aminoacid sequence identity with the parent antibody heavy or light chainvariable domain sequences, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, and most preferably at least95%. Identity or homology with respect to this sequence is definedherein as the percentage of amino acid residues in the candidatesequence that are identical with the parent antibody residues, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. None of N-terminal, C-terminal,or internal extensions, deletions, or insertions into the antibodysequence shall be construed as affecting sequence identity or homology.The variant retains the ability to bind the receptor and preferably hasproperties which are superior to those of the parent antibody. Forexample, the variant may have a stronger binding affinity, enhancedability to activate the receptor, etc. To analyze such properties, oneshould compare a Fab form of the variant to a Fab form of the parentantibody or a full length form of the variant to a full length form ofthe parent antibody, for example, since it has been found that theformat of the antibody impacts its activity in the biological activityassays disclosed herein. The variant antibody of particular interestherein is one which displays at least about 10 fold, preferably at leastabout 20 fold, and most preferably at least about 50 fold, enhancementin biological activity when compared to the parent antibody.

The “parent” antibody herein is one which is encoded by an amino acidsequence used for the preparation of the variant. Preferably, the parentantibody has a human framework region and has human antibody constantregion(s). For example, the parent antibody may be a human antibody intowhich the CDRs of a donor (murine) antibody are embedded.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The phrase “substantially free of cellular material” includespreparations of an antibody or antibody fragment in which the antibodyor antibody fragment is separated from cellular components of the cellsfrom which it is isolated or recombinantly produced. Thus, an antibodyor antibody fragment that is substantially free of cellular materialincludes preparations of antibody or antibody fragment having less thanabout 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (alsoreferred to herein as a “contaminating protein”). When the antibody orantibody fragment is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, or 5% of the volume of the proteinpreparation. When the antikine antibody or its antibody fragment isproduced by chemical synthesis, preferably it is produced free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the antibodyor antibody fragment have less than about 30%, 20%, 10%, 5% (by dryweight) of chemical precursors or compounds other than the antibody orantibody fragment of interest. In a one embodiment, antibodies of theinvention or fragments thereof are isolated or purified.

The term “antikine antibody” refers to an antibody as defined above thatbinds to two or more chemokines, preferably the antikine antibody willbind three or more human CC chemokines. An antikine antibody may be amonoclonal or polyclonal antibody, and preferably will be an isolated orpurified monospecific or monoclonal antibody. An antikine antibody maybe a mammalian antibody, such as a murine or human antibody, or achimeric or humanized antibody. Fully human antibodies may be obtainedfrom humans or from transgenic animals or phage display platforms, seeLonberg, Curr. Opin. Immunol. 20(4):450-9 (2008) to which procedures ofobtaining human antibodies from transgenic animals is incorporated byreference. Antikine antibodies may be synthetic antibodies, singledomain antibodies, such as nanobodies (V_(H)H) or camelized antibodies,single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab′)fragments, disulfide-linked Fvs, intrabodies, and anti-idiotypic(anti-Id) antibodies (including anti-idiotype and anti-anti-idiotypeantibodies to the antikine antibodies of the invention, such as 3C12F,etc.), bispecific, and fragments of any of the above that bind todeterminants or epitopes of a CC chemokine. Various structural forms ofengineered antibodies are incorporated by reference to AntibodyEngineering: A Practical Approach, edited by McCafferty, et al., OxfordUniversity Press (1996). The term “antikine antibody” encompassesimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules containing at least one antigen binding site(ABS). These may be of any isotype including IgA, IgD, IgE, IgG, IgM,and IgY and may be derived from vertebrates, such as mammals and birds,which produce antibodies. Preferably, antikine antibodies will be humanor humanized, though antikine antibodies may be suitable foradministration to animals, such as domestic or commercially raisedanimals, or wild animals or animals raised in captivity. These includecompanion animals, such as dogs and cats; livestock, such as bovine,equine, buffalo, water buffalo, swine, goats, sheep, camels, llamas,etc.; and fowl, such as chickens, turkeys, geese, falcons, etc. Those ofskill in the art would understand how to produce and adapt antikineantibodies for non-human uses, e.g., by inducing antibodies to CCchemokines expressed by a particular type of animal and/or by anantibody-engineering process analogous to humanization.

Antikine antibodies or fragments thereof will bind to specific CCchemokines and not non-specifically bind to other chemokines orpolypeptides. Antikine antibodies or their fragments thatimmunospecifically bind to a CC chemokine or a fragment of a CCchemokine may cross-react with other antigens. However, antikineantibodies or fragments that immunospecifically bind to a CC chemokineor fragment thereof may be selected that do not cross-react with otherantigens. Antikine antibodies or their fragments that immunospecificallybind to specific CC chemokines can be identified, for example, byimmunoassays or other techniques known to those of skill in the art.

A “fragment” describes a portion of an intact polypeptide molecule, suchas a CC chemokine or an antikine immunoglobulin. A “fragment” mayencompass a peptide or polypeptide comprising an amino acid sequence ofat least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100,120, 130, 150, 175, 200, or 250 contiguous amino acid residues of anamino acid sequence, such as the sequence of an intact mature CCchemokine or of a light or heavy antibody polypeptide chain. For a CCchemokine, a fragment will be a portion of the molecule that is shorterthan the length of the mature chemokine, such as a CC chemokine fragmentcomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, or any intermediate value up to but not including the fullamino acid sequence of the mature CC chemokine. For an antikineantibody, a fragment includes a peptide or polypeptide comprising anamino acid of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50contiguous amino acid residues, or up to but not including the fulllength of a V_(H) and/or V_(L) portion of an antibody that specificallybinds to a CC chemokine, and which binds to at least two or three CCchemokines bound by the intact antikine antibody. An antikine antibodyfragment may be a single chain fragment, e.g., a light or heavy chain ora portion of a light or heavy chain, but also includes fragments withmultiple chains, such as a Fab or F(ab)₂ fragments.

“Affinity matured antibodies” are antibodies that have had their bindingaffinity and/or biological activity increased by altering the type orlocation of one or more residues in the variable region. An example ofalteration is a mutation which may be in either a CDR or a frameworkregion. An affinity matured antibody will typically have its bindingaffinity increased above that of the isolated or natural antibody orfragment thereof by from 2 to 500 fold. Affinity matured antibodies mayhave nanomolar or even picomolar affinities to the receptor antigen.Affinity matured antibodies are produced by procedures known in the art.Marks, J. D. et al., Bio/Technology 10:779-783 (1992), which isincorporated by reference, describes affinity maturation by V_(H) andV_(L) domain shuffling. Random mutagenesis of CDR and/or frameworkresidues are incorporated by reference to Barbas, C. F. et al. Proc Nat.Acad. Sci, USA 91:3809-3813 (1994), Schier, R. et al. Gene 169:147-155(1995), Yelton, D. E. et al. J. Immunol. 155; 1994-2004 (1995), Jackson,J. R. et al. J. Immunol. 154(7):3310-9 (1995), and Hawkins, R. E. etal., J. Mol. Biol. 226:889-896 (1992).

An “antikine antibody derivative” refers to a polypeptide that comprisesan amino acid sequence of an antikine antibody or a CC chemokine bindingantibody fragment that specifically binds to at least two, three, fouror more CC chemokines, which has been altered by the introduction ofamino acid residue substitutions, deletions or additions. The term“derivative” as used herein also refers to an antikine antibody or afragment of an antikine antibody that has been covalently modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. Such an antikine antibody derivative may be produced by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. An antikineantibody derivative will retain an ability to specifically bind to twoor more CC chemokines.

An “antikine antibody analog” refers to a polypeptide comprising asubstantially similar amino acid sequence as a known antikine antibodyand which retains the ability of the known antikine antibody to bind totwo or more CC chemokines. An analog may also contain 1, 2, 3, 5 or 10or more non-classical amino acids. A polypeptide that has a similaramino acid sequence to an antikine antibody polypeptide may be describedby reference to identity to another protein or by reference to anencoding polynucleotide sequence include as:

(i) a polypeptide having an amino acid sequence that is at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or at least 99%identical to the amino acid sequence of at least one light or heavychain, or at least one CDR of a known antikine antibody;

(ii) a polypeptide encoded by a polynucleotide sequence that hybridizesunder stringent conditions to a nucleotide sequence encoding an antikineantibody, or antikine antibody fragment described herein, whichpolypeptide comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,90, 100, 125, or at least 150 amino acid residues. The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. Low stringency hybridization conditions, corresponding toa T_(m) of 55° C., include e.g., 5×SSC, 0.1% SDS, 0.25% milk, and noformamide; or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringencyhybridization conditions correspond to a higher T_(m), e.g., 40%formamide, with 5× or 6×SSC and high stringency hybridization conditionscorrespond to the highest T_(m), e.g., 50% formamide, 0.1×SSC; and

(iii) a polypeptide encoded by a nucleotide sequence that is at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99%identical to the nucleotide sequence encoding a known antikine antibody,or antikine antibody fragment. A polypeptide with similar structure toan antikine antibody, or antikine antibody fragment described hereinrefers to a polypeptide that has a similar secondary, tertiary orquaternary structure to the known antikine antibody of its fragment.Polypeptide and protein structure may be determined by methods known tothose skilled in the art, including but not limited to, X-raycrystallography, nuclear magnetic resonance, and crystallographicelectron microscopy.

The term “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” in the heavy chain and light chain variable domains;Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991) and/or those residues from a “hypervariable loop” in the heavychain and light chain variable domains; Chothia and Lesk, J. Mol. Biol.196:901-917 (1987). “Framework Region” or “FR” residues are thosevariable domain residues other than the hypervariable region residues asherein defined. Structural features of antibodies and methods fordetermining or analyzing antibody structure are well-known to those inthe art are incorporated by reference to Kabat, id., Chothia, et al.,id.; Déret, et al., Comput. Appl. Biosci. 11(4):435-9 (1995); Martin,Protein 25(1):130-3 (1996) and Abhinandan, et al., Mol. Immunol.45(14):3832-9 (2008); and Abhinandan, et al., J. Mol. Biol.369(3):852-62 (2007).

A “CC-chemokine” refers to a polypeptide from the family of chemotacticcytokines containing four conserved cysteine residues, the first two ofwhich are adjacent as described for example by Van Coillie, et al.,Cytokine & Growth Factor Rev. 10:61-86 (1999). CC chemokines are alsoknown as β-chemokines. Based on these adjacent cysteine-cysteine(cys-cys, or C—C) residues the β-chemokines are known as “CC”chemokines, where “CC” denotes the adjacent cysteine residues. Examplesof CC chemokines are those binding to CCR1 and CCR5, includingCCL3/MIP-1α, CCL4/MIP-1β and CCL5/RANTES. CC chemokines exist in mammalsand birds. Terms used for the human or murine version of a particularchemokine, e.g., human CCL3/MIP-1α may be used to identify thecorresponding molecule in another species, e.g., “murine MIP-1α” or“bovine MIP-1α” and should be understood to refer to the analogous,structurally similar molecule in the species being referred to.Analogous chemokines among mammalian and avian species may have at least40%, 50%, 60%,70%, 80%, 90%, 95% or 99%, or any intermediate valuewithin this range, sequence identity and will exhibit the same orsimilar functional immunological activities.

Sequences for vertebrate CC chemokines, including those of mammals andbirds, are incorporated by reference to the NCBI database with specificreference to those sequences identified by the last updated accessionnumbers in that database (last accessed on Aug. 9, 2010) and toFernandez & Lolis, Annu Rev. Pharmacol. Toxicol. 42:469 (2002) which ishereby incorporated by reference.

The specific accession numbers for the polynucleotide and polypeptidesequences of human chemokines CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β,CCL5/RANTES, CCL14/HCC-1, CCL15/HCC-2, CCL18/PARC, and CCL23/MPIF-1 andothers are provided in Table 4. Each is specifically incorporated byreference to the catalog, supplier, or NCBI accession numbers describedin Table 4. These chemokines are commercially available from thesuppliers described in Table 4.

The term “CC chemokine determinant” as used herein refers to portions ofa CC chemokine which are contacted by the antigen binding residues of anantibody. This term encompasses both conventional linear epitopesrecognized by an antikine antibody on a CC chemokine as well asconformational epitopes, including nonlinear epitopes. The one or moreportions of a CC chemokine bound by an antikine antibody or directly incontact with its antigen binding amino acid residues are itsdeterminants. Removal, replacement, disruption or denaturation of anantigenic determinant of a CC chemokine may reduce or eliminate theability of an antikine antibody to bind to the CC chemokine. Methods fordetermining antibody binding epitopes are well-known in the art as shownby Ladner, Biotechnol. Genet. Eng. Rev. 24:1-30 (2007), which isincorporated by reference. CC chemokine determinants include segments ordomains involved in binding to a chemokine receptor (e.g., CCR1 orCCR5), such as its N-loop, 2 and 3 strands, the 30's, 40's and 50'sloops and its C terminal helical segment, these determinants areincorporated by reference to Fernandez & Lolis, Annu. Rev. Pharmacol.Toxicol. 42:469 (2002), Viola, et al., Annu. Rev. Pharmacol. Toxicol.48:171, and Pakianathan, et al., Biochem. 36(32):9642 (1997).

CC chemokine determinants are also defined by binding of proteins frompathogens, such as the CC chemokine determinants bound by vCCI ofvaccinia virus, these determinants are incorporated by reference toZhang et al., Proc. Natl. Acad. Sci. 103(38):13985-13990 (2006).

The terms “bind” or “binding” refer to a chemical or physical contact orinteraction between two different substances. This includes anassociation between an antikine antibody and a correspondingdeterminant, such as an epitope on a CC chemokine molecule. Such contactor interaction may be by one or more of ionic, non-ionic, hydrogenbonding, van der Waals bonding, hydrophobic bonding, or other knowntypes of intermolecular bonding. Binding may involve direct contactbetween two molecules, such as between a ligand and its correspondingreceptor, or indirect through an intervening moiety, such as covalent ornoncovalent binding via a linker or bi- or polyvalent moiety.

“Binding affinity” is described by affinity constant Ka, which is theratio between the rate constants for binding and dissociation ofantibody and antigen. Typical affinities for IgG antibodies are10⁻¹⁰-10⁻⁹ mole/L. Antibody affinity is measured by many differenttechniques known in the art including: equilibrium dialysis, surfaceplasmon resonance (BIAcore®), and kinetic exclusion assay (KinExA®). Therelationship between bound and free antigen and antibody affinity isexpressed by the Scatchard equation, r/c=Kn−Kr, where r=the ratio of[bound antigen] to [total antibody], c=[free antigen], K=affinity, andn=number of binding sites per antibody molecule (valence). If all theantibodies have the same affinity for antigen (e.g., a monoclonalantibody), a plot of r/c versus r will yield a straight line with aslope of −K and an r intercept approaching n. If the antibody isheterogeneous (e.g., polyclonal), the plot of r/c versus r will yield acurved line; the average affinity can be determined by the slope of thecurve when half the binding sites are full (r=1).

The phrase “specifically binds to a CC chemokine” describes anabove-background interaction of antibody or an antibody fragment with aparticular CC chemokine or set of CC chemokines, but not with otherchemokines. CC chemokines include, but are not limited to, CCL3/MIP-1α,CCL4/MIP-10, and CCL5/RANTES, and examples of CC chemokine segments ordomains include its chemokine receptor binding residues, its N-loop,β-1, 2 and 3 strands, the 30's, 40's and 50's loops and its C terminalhelical segment. An antibody that specifically binds to a CC chemokineor one of its domains or segments may bind to other antigens with loweraffinity as determined by, e.g., immunoassays, surface plasmon resonance(BIAcore®), or other assays known in the art. For example, an antikineantibody that has a four-fold or greater ability to bind to a particularCC chemokine compared to another or control antibody when compared atthe same concentration and under the same conditions would be consideredto specifically bind to the CC chemokine. However, other comparativebinding criteria may be used which establish a significant difference inthe amount or affinity of binding, these would include a 2, 3, 4, 5, 10,20, 50, 100 or 1000-fold greater amount of binding for the antikineantibody or a 2, 5, 10, 15, 20, 100, or at least 1,000-fold greaterbinding affinity.

The antibodies or fragments that specifically bind to two, three, four,five, or more CC chemokines need not cross-react with othernon-chemokine antigens or non-CC chemokines. Antikine antibodies orfragments that specifically bind to CC chemokines or their fragments canbe identified by immunoassays, BIAcore, or other techniques known tothose of skill in the art. An antibody or a fragment thereof bindsspecifically to a CC chemokine antigen or fragment thereof with higheraffinity than to any cross-reactive antigen as determined usingexperimental techniques, such as Western blots, radioimmunoassays (RIA)and enzyme-linked immunosorbent assays (ELISA). See, e.g., Paul, ed.,Fundamental Immunology, Second Edition, Raven Press, New York (1989) atpages 332-336 for a discussion regarding antibody specificity.

The term “inhibitory concentration” refers to a concentration ofantibody, such as an antikine antibody or its CC chemokine-bindingfragments, which in comparison to a control reduce a CC chemokineactivity, for example by 5%, 10%, 20%, 30%, 50%, 80%, 90%, 95%, 99% orup to 100%. CC chemokine activity may be determined in vivo or in vitroand include assays of CC chemokine activities, such as chemotaxis. Suchassays include calcium flux or integrin activation. The modulatoryeffects of an antikine antibody or its CC-chemokine binding fragments,as well as control antibodies, such as those which do not bind to CCchemokines or those binding to a single chemokine, may also bedetermined in in vivo animal models, including those measuring CCchemokine activation of immune phenomena, such as inflammation andautoimmunity. Inhibiting the activity of a chemokine refers to causing arelative decrease in at least one activity of a chemokine in thepresence of an inhibitory agent as compared to the activity in theabsence of the agent. Inhibition may involve antagonism orneutralization of chemokine activity, for example, by antibody bindingto an active site on the chemokine, or by binding that leads toeffective removal, immobilization, or inactivity of the chemokine. Theterm “chemotaxis inhibition” refers to a decrease in the relative amountof chemotactic activity of cells in the presence of the antibody orantigen-binding fragment thereof in comparison with chemotactic activityobserved in the absence of the antibody or antigen-binding fragmentthereof. Well known methods of measuring chemotactic inhibition ofparticular types of cells, including different leukocyte cell types, areavailable and are incorporated by reference to Chemokine Protocols,Meth. in Mol. Biol. 138 (2000), Humana Press, Eds. AEI Proudfoot, TNCWells, and CA Power.

An “isolated” or “purified” product or component is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the product or component is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. An isolated or purified component, molecule orother substance (including a peptide, polypeptide, antibody, chemokine,polynucleic acid, or cell) is one that has been removed from, orsynthesized separately from, its ordinary or natural or indigenousenvironment. An isolated or purified product or component may also bephysically or chemically removed or separated from the admixture oringredients with which it is associated, including undesired biologicalcontaminants, or, if synthesized, by substrates or byproducts associatedwith its synthesis. Purification may extend to any degree includingremoval of 1, 5, 10, 50, 75, 90, 95 or 100% of the other components. Inthe case of an antibody, isolation may constitute removal from blood orserum, or for a monoclonal antibody, from ascites or tissue culturefluid.

The terms “nucleic acids” and “nucleotide sequences” include DNAmolecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA),combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, andanalogs of DNA or RNA molecules. The nucleic acid sequences of theinvention may encode portions of an antikine antibody analog or variant.Such analogs can be generated using, for example, nucleotide analogs,which include, but are not limited to, inosine or tritylated bases. Suchanalogs can also comprise DNA or RNA molecules comprising modifiedbackbones that lend beneficial attributes to the molecules such as, forexample, nuclease resistance or an increased ability to cross cellularmembranes. The nucleic acids or nucleotide sequences can besingle-stranded, double-stranded, may contain both single-stranded anddouble-stranded portions, and may contain triple-stranded portions, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid molecule. An“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. In a oneembodiment, nucleic acid molecules encoding antibodies of the inventionor fragments thereof are isolated or purified.

The term “host cell” as used herein refers to the particular subjectcell transfected with a nucleic acid molecule and the progeny orpotential progeny of such a cell. Progeny of such a cell may not beidentical to the parent cell transfected with the nucleic acid moleculedue to mutations or environmental influences that may occur insucceeding generations or integration of the nucleic acid molecule intothe host cell genome. A host cell may be used to recombinantly expressan antikine antibody or one of its components, e.g., a light or heavychain or fragment thereof.

To determine the “percent identity” of two nucleic acid or amino acidsequences, the sequences are first aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions multipliedby 100%). In one embodiment, the two sequences are the same length. Thedetermination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad.Sci. U.S.A. 87:2264-2268 (1990), modified as in Karlin and Altschul,Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877 (1993). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al., J.Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performedwith the NBLAST nucleotide program parameters set, e.g., for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecule of the present invention. BLAST protein searches can beperformed with the XBLAST program parameters set, e.g., to score=50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Alternatively,PSI-BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, CABIOS4:11-17 (1988). Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM 120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

A “conservative change” refers to alterations that are substantiallyconformationally or antigenically neutral; producing minimal changes inthe tertiary structure of a peptide or polypeptide variant, or producingminimal changes in the antigenic determinants of the variant oranalogous peptides or polypeptides, as compared to the parental ornative peptide or polypeptide. In the context of chemokines,conservative changes include amino acid substitutions that do notsubstantially affect the specificity and/or affinity of the resultingchemokine variant or analog, for example, as determined by its abilityto exhibit at least one function of the parental chemokine includingsuch functions as induction of chemotaxis, participation in cytokinenetworks or otherwise inducing enzyme or cytokine production, or bindingto the receptor for the parental or native chemokine. As applied tovariants or analogs of the antibodies, antibody fragments, including CDRsegments, a conservative change refers to an amino acid substitutionthat produces an antibody product that is able to bind to the sameepitope or antigen as the corresponding unmodified antibody product.Prediction of which amino acid substitutions maintain the conformationaland antigenic neutrality of a molecule are within the skill of the artas described by Berzofsky, Science 229:932-940 (1985) and Bowie, et al.,Science 247:1306-1310 (1990). Guidance as to which substitutions willmost likely maintain conformational and antigenic neutrality include (a)substitution of hydrophobic amino acids is less likely to affectantigenicity because hydrophobic residues are more likely to be locatedin a protein's interior, (b) substitution of physiochemically similaramino acids is less likely to affect conformation because thesubstituted amino acid structurally mimics the native amino acid; and(c) alteration of evolutionarily conserved sequences is likely adverselyto affect conformation as such conservation suggests that the amino acidsequences may have functional importance. A “composition” or“pharmaceutical or therapeutic composition” refers to a combination ofcarrier, excipient, or solution containing an antikine antibody or itsCC-chemokine binding fragments, or its other fragments, that directly orindirectly reduce the severity of or treat a condition, disorder ordisease mediated by a CC chemokine, or at least one symptom thereof. Theterm “pharmaceutically acceptable carrier” includes any and all carriersand excipients such as diluents, solvents, dispersing agents, emulsions,lipid bilayers, liposomes, coatings, preservatives includingantibacterial or antifungal agents, isotonic agents, pH buffers, andabsorption modulating agents, and the like, compatible with themolecules of the present invention and suitable for pharmaceuticaladministration. The use of such carriers, disintegrants, excipients andagents for administration of pharmaceutically active substances is wellknown in the art, see the Handbook of Pharmaceutical Excipients, 3^(rd)edition, Am. Pharm. Assoc. (2000) which is incorporated by reference.The pharmaceutical compositions of the invention are generallyformulated for compatibility with an intended route of administration,such as for parenteral, oral, or topical administration.

The therapeutic compositions of the invention include at least oneantibody or antibody fragment of the invention in a pharmaceuticallyacceptable carrier. A “pharmaceutically acceptable carrier” will be atleast one component conventionally admixed with, and used for, theadministration of an active ingredient, biological product, or drug. Acarrier may contain any pharmaceutical excipient used in the art and anyform of vehicle for administration. The compositions may be, forexample, injectable solutions, aqueous suspensions or solutions,non-aqueous suspensions or solutions, sprays, solid and liquid oralformulations, salves, gels, ointments, intradermal patches, creams,lotions, tablets, capsules, sustained release formulations, and thelike. Additional excipients may include, for example, colorants,taste-masking agents, solubility aids, suspension agents, compressingagents, enteric coatings, sustained release aids, and the like. Asuitable dosage form may be selected by one of skill in the art fromforms such as those described by the U.S. FDA CDER Data Standards ManualC-DRG-00201, Version 08; or those listed at the FDA websitehttp://www.fda.gov/ForIndustry/DataStandards/StructuredProductLabeling/ucm162038.htm(last accessed Aug. 9, 2010); both of which are hereby incorporated byreference.

Orally administered compositions can include a solid carrier orexcipient or may be formulated as liquid or gel preparations and mayinclude an edible or inert carrier and may be enclosed in capsules,compressed into tablets, or formulated as a troche. Orally administeredcompositions may be prepared in a time-release or encapsulated form toprevent degradation in the stomach and optimize uptake of a molecule.

Injectable compositions may be formulated by methods well known in theart and may encompass sterile solutions or dispersions of therapeuticmolecules. Such will usually include a sterile diluent, such as water,normal saline, or other buffer compatible with the molecules of theinvention. Injectable compositions may be prepared in unit dosages or inunit dose containers, such as vials, ampules, or syringes.

Conventional buffers and isotonic agents may be used and pH may beadjusted using well known agents, such as HCl or NaOH or buffers.Antimicrobial or bacteriostatic agents, chelating agents, such as EDTAor EGTA, and antioxidants and preservatives may be present.

The therapeutic compositions of the invention may be administered by anyacceptable route of administration including topically, on to a mucousmembrane, orally or enterically or parenterally. These routes include,but not limited to topical, transmucosal, orally (including buccal,sublingual), mucosally (conjunctiva, nasal, sinal, urethral, vaginal,intestinal, rectal), enteric, transdermal, intradermal, subcutaneous(s.c.), intramuscular, intraperitoneal, intravenous (i.v.) intracardiac,into a joint or bone, into an organ (brain, spinal chord, eye, ear,liver, spleen, kidney, gall bladder, bladder), into bone, cartilage, orjoint tissue, by inhalation (e.g., intranasal, intratracheal,intrapulmonary, or intrabroncial), oral, subuccal. Routes may beselected by those of skill in the art from those listed in the U.S. FDA,CDER, Data Standards Manual “Routes of Administration”, CDRG-00301,Version 004; Data Element Name. Route of Administration.

Data Element OID: 2.16.840.1.113883.3.26.1.1.1 Data Element NCI ConceptID: C38114

Version Number. 004

available athttp://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/DrugRegistrationandListing/ucm084039.htm(last accessed Aug. 9, 2010); which are hereby incorporated byreference.

A “therapeutically effective amount” refers to that amount of thetherapeutic agent sufficient to reduce the severity of or treat acondition, disorder or disease mediated by CC chemokines, to enhance thetherapeutic efficacy of another therapy of the condition, disorder ordisease, or to prevent the recurrence or an increase in severity of thecondition, disorder or disease or at least one of its symptoms. Atherapeutically effective amount may refer to the amount of therapeuticagent sufficient to delay or minimize the onset of disease. Atherapeutically effective amount may also refer to the amount of thetherapeutic agent that provides a therapeutic benefit in the treatmentor management of a disease. Further, a therapeutically effective amountwith respect to a therapeutic agent of the invention means that amountof therapeutic agent alone, or in combination with other therapies, thatprovides a therapeutic benefit in the treatment or management of adisease, e.g., sufficient to enhance the therapeutic efficacy of atherapeutic antibody sufficient to treat or manage a disease. Used inconnection with an amount of an antibody of the invention, the term canencompass an amount that improves overall therapy, reduces or avoidsunwanted effects, or additively enhances the therapeutic efficacy of, orsynergizes with, another therapeutic agent.

The term “subject” or “patient” as used herein refers to a vertebratethat expresses CC chemokines, including avian and mammals (e.g.,bovines, equines, swine, goats, sheep, canines, or felines) andpreferably a human. A subject or patient may be one in need of treatmentwith an effective amount of an antikine antibody that modulates CCchemokine activity of the CC chemokines it recognizes.

An “inflammatory condition, disorder, or disease” refers to an evidentor quantifiable physiological phenomenon, including, but not limited toedema, fever, chemotaxis or migration of leukocytes, proliferation ofblood vessels, proliferation of connective tissue, redness, localizedheat, exudation, and other signs as described in Robbins, ThePathological Basis of Disease, 6^(th) edition, Cotran, et al. (eds.),W.B. Saunders, Co. (1999), especially Chapters 3, 7 and 15. In thecontext of antikine antibodies to CC-chemokines, this term would referto phenomena associated with, or directly or indirectly mediated by, aCC-chemokine, such as MIP-1α, RANTES, MCP-1β, or other chemokines.

An “immunoaffinity resin” refers to a solid substrate to which at leastone immunological ligand or receptor is bound. For example, an antikineantibody may be bound to a resin via a region other than its antigencombining site, thereby allowing the antibody to bind antigenicdeterminants on CC chemokines. Similarly, a CC chemokine may beoperatively bound to a resin permitting it to bind to an antikineantibody. Many resins useful for immobilizing immunological ligands orreceptors are known in the art and are routinely used to formimmunoaffinity resins. Such resins are useful in analysis of componentsbound by a particular ligand or receptor, in immunological assays or inpurification procedures. Any such resin may be used to form theimmunoaffinity resins of the invention.

The inventors pursued means for solving the prior art problemsassociated with administering multiple antibodies to CC chemokines.Surprisingly, they discovered antibodies which can bind to more than onetype of CC chemokine: antikine antibodies. These antikine antibodiesprovide improvements over inhibiting a single chemokine or a singlechemokine receptor in an inflammatory disease setting since only asingle antibody is needed to block the functions of multiple chemokines.The antikine antibodies of the invention also avoid the drawbacks ofadministering two or more antibodies having different pharmacokineticproperties, such as use of inconvenient separate dosing regimens foreach antibody. Antikines avoid the disadvantages of bispecificantibodies which are less effective at binding to a specific chemokineantigen and form complexes which bind more readily to Fc receptors andthus taken up by cells and degraded in lysosomes.

As demonstrated herein, the inventors provide several antikineantibodies that can simplify treatment of human inflammatory andimmunological conditions, disorders and diseases mediated by multiplechemokines. These antikine antibodies will serve as structural andfunctional prototypes for producing even more effective and safeantikine antibodies by processes such as affinity maturation andantibody humanization.

Antikine antibodies bind to and inhibit the activities of two or more CCchemokines. By targeting CC chemokines, including MIP-1α, MIP-1β, RANTESand MCP-1, the inventors identified antibodies useful for modulatingchemokines activating the same or different chemokine receptors(including CCR1, CCR2, CCR3, and CCR5). For example, since MIP-1α,MIP-1β, and RANTES each bind to chemokine receptor CCR5, an antikineantibody which recognizes these three CC chemokines can morecomprehensively modulate CCR5 activity than one to a single CCchemokine. CCR5 activation targets immature myeloid dendritic cells,monocytes, Th 1, L_(reg), NK and plasmacytoid dendritic cells.Similarly, MIP-1α, RANTES, MPIF-1 and HCC-1 each bind to CCR1, whichactivation targets monocytes, memory T cells and NK cells. An antikineantibody binding to two or more of these CCR1-binding chemokines morecomprehensively modulates receptor activation. An antibody that can bindand inhibit both MIP-1α and RANTES would then effectively block two ofthe primary ligands of both CCR1 and CCR5. Thus, the inventors haveidentified antikine antibodies that inhibit the primary ligands fordifferent chemokine receptors expressed on leukocytes includingmonocytes and T cells implicated in inflammatory diseases. Inhibitors ofeither of these combinations of chemokines provide improvements overinhibiting a single chemokine (or antibodies that block binding to asingle chemokine receptor) in an inflammatory disease setting since onlya single antibody is needed to block the functions of multiplechemokines on a single receptor or chemokine activity on multiplereceptors.

Antikine antibodies have different specificities and functionalactivities depending on the chemokines to which they bind. The inventionprovides antikine antibodies that specifically bind to a variety ofCC-chemokines, and advantageously to at least two of RANTES, MIP-1α,MIP-1β and/or MCP-1. The invention further provides antibodies thatspecifically bind to both RANTES and MIP-1α; RANTES and MIP-1β; RANTESand MCP-1; MIP-1α and MIP-1β; MIP-1α and MCP-1; or MIP-1β and MCP-1.Similarly, the invention provides antikine antibodies that bind to threeor four of RANTES, MIP-1α, MIP-1β and/or MCP-1 as well as to otherclosely related CC-chemokines.

The binding of an antibody or antigen-binding antibody fragment to achemokine occurs by contact between the amino acid residues in theantigen binding sites (ABS) of the antibody and the antigenicdeterminants or epitopes of the chemokine. It is well-known thatepitopes may be linear peptide sequences or conformation epitopescomprising multiple antigenic determinants, even single amino acids oramino acid side groups which do not form part of a linearly contiguousamino acid sequence. For example, the residues lying on one face of anα-helix may be contacted by the ABS of an antibody, while those on theopposite face are not.

The antibodies and antigen-binding antibody fragments of the inventionmay bind to the N-terminal or C-terminal half of a mature CC chemokinewhich does not contain a signal peptide. For example, the mature form ofhuman RANTES lacks signal peptide residues 1-23. Thus, its N-terminalportion would range from residue 1-35 and its C-terminal would rangefrom 36-68 (SEQ ID NO: 73).

The antibodies and antigen-binding antibody fragments of the inventionmay block CC chemokine binding by binding to CC chemokine residuesassociated with binding of the chemokine to its receptor. See NCBIConserved Domain Database CDD 29111 [uid] for proposed functionaldomains of CC chemokines; http://www.ncbi.nlm.nih.gov/sites/entrez (lastaccessed Aug. 9, 2010). Prospective binding site residues are indicatedfor each of the CC chemokines below:

CCL3/MIP-1α residues 11-15 (CCFSY), residues 17-24 (SRQIPQNF), residues34-35 (QC), or residues 57-67 (EWVQKYVSDLE) of SEQ ID NO: 71;

CCL4/MIP-1β residues 11-15 (CCFSY), residues 17-24 (ARKLPHNF), residues34-35 (LC), or residues 57-67 (SWVQEYVYDLE) of SEQ ID NO: 72;

CCL5/RANTES residues 10-14 (CCFAY), residues 16-23 (ARPLPRAH), residues33-34 (KC), or residues 56-66 (KWVREYINSLE) of SEQ ID NO: 73.

CCL14/HCC-1 residues 8-12 (CCFTY), residues 14-21 (TYKIPRQR), residues31-32 (QC), or residues 54-64 (KWVQDYIKDMK) of SEQ ID NO: 78.

CCL15/HCC-2 residues 8-12 (CCTSY), residues 14-21 (SQSIPCSL), residues31-32

(EC), or residues 54-64 (PGVQDCMKKLK) of SEQ ID NO: 79.

CCL23/MPIF-1 residues 9-13 (CCISY), residues 15-22 (PRSIPCSL), residues32-33 (EC), or residues 55-65 (KQVQVCMRMLK) of SEQ ID NO: 81.

CCL18/PARC residues 10-14 (CCLVY), residues 16-23 (SWQIPQKF), residues33-34 (QC), or residues 56-66 (KWVQKYISDLK) of SEQ ID NO: 82.

Such an antibody or antigen binding fragment may bind to segments of atleast two of MCP-1, MIP-1α, MIP-1β and RANTES, as well as other relatedCC-chemokines, involved in binding of said chemokine to its receptor.Antikines, such as 3C12F, may bind to the same determinants aschemokine-binding viral proteins such as vCCI, or may inhibit thebinding of such viral proteins to chemokines.

Monoclonal antibodies 3C12F, 7D1G, 7D12A, 18V4F and 18P7E which bind theCC-chemokines RANTES, MIP-1α and/or MIP-1β have been produced andcharacterized by the inventors. These antibodies provide necessaryinformation, including structural information from hypervariable regionsand light and heavy chain CDRs, for developing humanized antibodies thatcan simultaneously modulate the activity of multiple CC chemokines andfor the treatment of inflammatory diseases mediated by CC chemokines.The hypervariable and CDR sequences of the three MAbs above are depictedby SEQ ID NOS: 2-5 and 7-10 for 3C12F; SEQ ID NOS: 52-55 and 57-60 forhumanized 3C12F; SEQ ID NOS: 12-15 and 17-20 for 7D12A; SEQ ID NOS:22-25 and 27-30 for 7D1G SEQ ID NOS: 32-35 and 37-40 for 18V4F, SEQ IDNOS: 62-65 and 67-70 for humanized 18V4F; and SEQ ID NOS: 42-45 and47-50 for 18P7E.

Five specific types of antikine antibodies characterized by the specificantikine antibodies 3C12F, 7D1G, 7D12A, 18V4F and 18P7E are disclosed.In addition, humanized versions of 3C12F and 18V4F are disclosed.

The first type of antibody is characterized by the binding specificityof monoclonal antibodies made by hybridoma cell line 3C12F or asubculture thereof. A typical antibody of this type of monoclonalantibody (MAb) is 3C12F. Thus this type includes MAb 3C12F, as well asits analogs and derivatives, including those antibodies produced by theprocess of affinity maturation or by humanization. Antibodies of the3C12F type may contain a heavy chain variable region depicted by SEQ IDNO: 2 or 52 or a heavy chain containing at least one of CDR1 (SEQ ID NO:3 or 53), CDR2 (SEQ ID NO: 4 or 54) and CDR3 (SEQ ID NO: 5 or 55) of the3C12F heavy chain. Such antibodies can contain a light chain variableregion comprising SEQ ID NO: 7 or 57 or containing at least one of CDR1of SEQ ID NO: 8 or 58, CDR2 as of SEQ ID NO: 9 or 59, and CDR3 as setforth by SEQ ID NO: 10 or 60. SEQ ID NOS: 2-5 and 7-10 describenon-humanized 3C12F, while SEQ ID NOS: 52-55 and 57-60 describehumanized 3C12F. Humanized 3C12F contains a heavy chain variable regiondepicted by SEQ ID NO: 52 or a heavy chain containing at least one ofCDR1 (SEQ ID NO: 53), CDR2 (SEQ ID NO: 54) and CDR3 (SEQ ID NO: 55) ofthe 3C12F heavy chain. Such antibodies can contain a light chainvariable region comprising SEQ ID NO: 57 or containing at least one ofCDR1 of SEQ ID NO: 58, CDR2 as of SEQ ID NO: 59, and CDR3 as set forthby SEQ ID NO: 60.

A second type of antibody is characterized by the binding specificity ofmonoclonal antibodies made by hybridoma cell line 7D12A or a subculturethereof. A typical antibody of this type of monoclonal antibody (MAb) is7D12A. Thus, this type includes MAb 7D12A, as well as its analogs andderivatives, including those antibodies produced by the process ofaffinity maturation or by humanization. Antibodies of the 7D12A antibodytype may contain a heavy chain variable region depicted by SEQ ID NO: 12or a heavy chain containing at least one of CDR1 (SEQ ID NO: 13), CDR2(SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the 7D12A heavy chain. Theymay also contain a light chain variable region comprising SEQ ID NO: 17or containing at least one of CDR1 of SEQ ID NO: 18, CDR2 as of SEQ IDNO: 19, and CDR3 as set forth by SEQ ID NO: 20.

A third type of antibody is characterized by the binding specificity ofmonoclonal antibodies made by hybridoma cell line 7D1G or a subculturethereof. A typical antibody of this type of monoclonal antibody (MAb) is7D1G. This type includes MAb 7D1G and its analogs and derivatives,including those antibodies produced by the process of affinitymaturation or by humanization. The 7D1G type of antibody may containheavy chain variable region depicted by SEQ ID NO: 22 or a heavy chaincontaining at least one of CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO: 24)and CDR3 (SEQ ID NO: 25) of the 7D1G heavy chain. They may also containa light chain variable region comprising SEQ ID NO: 27 or containing atleast one of CDR1 of SEQ ID NO: 28, CDR2 as of SEQ ID NO: 29, and CDR3as set forth by SEQ ID NO: 30.

A fourth type of antibody is characterized by the binding specificity ofmonoclonal antibodies made by hybridoma cell line 18V4F or a subculturethereof. A typical antibody of this type of monoclonal antibody (MAb) is18V4F. This type includes MAb 18V4F and its analogs and derivatives,including those antibodies produced by the process of affinitymaturation or by humanization. The 18V4F type of antibody may containheavy chain variable region depicted by SEQ ID NO: 32 or 62 or a heavychain containing at least one of CDR1 (SEQ ID NO: 33 or 63), CDR2 (SEQID NO: 34 or 64) and CDR3 (SEQ ID NO: 35 or 65) of the 18V4F heavychain. They may also contain a light chain variable region comprisingSEQ ID NO: 37 or 67 or containing at least one of CDR1 of SEQ ID NO: 38or 68, CDR2 as of SEQ ID NO: 39 or 69, and CDR3 as set forth by SEQ IDNO: 40 or 70. SEQ ID NOS: 32-35 and 37-40 describe non-humanized 18V4F,while SEQ ID NOS: 62-65 and 67-70 describe humanized 18V4F. Humanized18V4F contains a heavy chain variable region depicted by SEQ ID NO: 62or a heavy chain containing at least one of CDR1 (SEQ ID NO: 63), CDR2(SEQ ID NO: 64) and CDR3 (SEQ ID NO: 65) of the 18V4F heavy chain. Suchantibodies can contain a light chain variable region comprising SEQ IDNO: 67 or containing at least one of CDR1 of SEQ ID NO: 68, CDR2 as ofSEQ ID NO: 69, and CDR3 as set forth by SEQ ID NO: 70.

A fifth type of antibody is characterized by the binding specificity ofmonoclonal antibodies made by hybridoma cell line 18P7E or a subculturethereof. A typical antibody of this type of monoclonal antibody (MAb) is18P7E. This type includes MAb 18P7E and its analogs and derivatives,including those antibodies produced by the process of affinitymaturation or by humanization. The 18P7E type of antibody may containheavy chain variable region depicted by SEQ ID NO: 42 or a heavy chaincontaining at least one of CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 44)and CDR3 (SEQ ID NO:45) of the 18P7E heavy chain. They may also containa light chain variable region comprising SEQ ID NO: 47 or containing atleast one of CDR1 of SEQ ID NO: 48, CDR2 as of SEQ ID NO: 49, and CDR3as set forth by SEQ ID NO: 50.

In addition to the specific types of antikine antibodies describedabove, the invention encompasses antikine antibodies that bind to three,four, five or more CC-chemokines, for example, antikine antibodies, suchas MAb 3C12F and the others described above, that bind to MIP-1α,MIP-1β, RANTES and/or other related CC-chemokines. Advantageously,antikine antibodies may be produced to all the CC chemokines that bindto a particular CCR, or may be targeted to those CC chemokines involvedin a particular condition, disorder or disease.

An antikine antibody will have a binding affinity sufficient to permitit to bind to a CC chemokine. Exemplary binding affinities include1,000, 900, 800, 400, 200, 150, 100, 75, 50, 40, 30, 20, 10, 5, 1, 0.1nM or less for the CC chemokines it binds. However, it may havedifferent binding affinity for different CC chemokines as shown in theFigures.

Selection of an antibody having an appropriate binding affinity iswithin the skill of those in the art. For many therapeutic purposes anantibody with a high affinity is more desirable than one with a lowaffinity, for example, passively administered high affinity antibodieshave been demonstrated to more efficiently remove antigen in vivo thanlow affinity antibodies; higher affinity antibodies produced lowerlevels of circulating immune complexes and resulted in less impairmentof glomerular function; Steward, Antibodies: Their Structure andFunction, Taylor and Francis (1984), see Table 4.5, to which methods forantibody affinity determination as well as the characteristics andfunctions of low and high affinity antibodies is incorporated byreference. On the other hand, keeping in mind that the neutralizingpower of an antibody depends not only on its affinity, but also itsconcentration, valence and molecular configuration, as well as on thesite to which it is administered, for some applications a higherconcentration of a lower affinity antibody may be desirable.

Immunogens used to produce antikine antibodies may include a preparationof isolated, native CC-chemokines, recombinantly expressed CC-chemokinesor chemically synthesized CC-chemokines, and optionally, an adjuvant.Various adjuvants used to increase the immunological response include,but are not limited to, Freund's (complete and incomplete), mineral gels(e.g., aluminum hydroxide), surface active substances (e.g.,lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerinand Corynebacterium parvum, or similar immunostimulatory agents.

The sequences of MIP-1α, RANTES, and MCP-1 proteins and polynucleotidesencoding such proteins are known and may be found, for example, inpublicly available sequence databases such as GenBank or by reference tothe accession numbers in Table 4. Unless otherwise specified, thepertinent versions of these sequences will be those entered into thesedatabases immediately before the filing date of this application. Inaddition, the sequences of various CC-chemokines, including MIP-1α,MIP-1β, RANTES, and MCP-1, have been published, and may be found, forexample, in Furutani, et al., Biochem. Biophys. Res. Commun. 159:249-255(1989)(MCP-1), Obaru, et al., J. Biochem. 99:885-894 (1986) (MIP-1α),Lipes, et al., Proc. Natl. Acad. Sci. USA 85:9704-9708 (1988) (MIP-1β),Schall, et al., J. Immunol. 141:1018-1025 (R1988)(RANTES), thedisclosure of each of which is incorporated by reference herein in itsentirety. It is well known that allelic variants exist for some or allof these chemokines. SEQ ID NOS: 71-74 depict the human sequences usedfor immunizations and screening of antikine antibodies for MIP-1α,MIP-1β, RANTES, and MCP-1.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byinjection with the native protein, or a synthetic variant thereof, or aderivative of a CC chemokine or combination of more than one CCchemokine. For example, a cocktail of MIP-1α, MIP-1β, RANTES, and MCP-1may be used as an immunogen to induce an immune response against theseCC chemokines.

If desired, the polyclonal antibody molecules directed against the CCchemokines can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography, to obtain the immunoglobulin fraction and immunoaffinitypurification to select antikine antibodies binding to two or more CCchemokines.

For many applications it will be preferable to produce an antikineantibody as a monoclonal antibody. Any technique that provides for theproduction of antibody molecules by continuous cell line culture may beutilized. Such techniques include, but are not limited to, the hybridomatechnique—see Köhler & Milstein, Nature 256:495-497 (1975); the triomatechnique; the human B-cell hybridoma technique, see Kozbor, et al.,Immunol. Today 4:72 (1983) and the EBV hybridoma technique to producehuman monoclonal antibodies, see Cole et al., In: Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985). Humanmonoclonal antibodies may be utilized in the practice of the presentinvention and may be produced by using human hybridomas as described byCote, et al., Proc. Natl. Acad. Sci. USA 80: 2026-2030 (1983) or bytransforming human B-cells with Epstein Barr Virus in vitro, see Cole,et al. In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96 (1985). All of the documents cited in this paragraph areincorporated by reference.

Beyond these conventional ways to produce monoclonal antibodies, theinventors have discovered that sequential immunization methodssurprisingly, in view of the problem of original antigenic sin (theHoskins Effect), produce antikine antibodies recognizing more than oneCC chemokine. The sequence of immunization was found to influence thespecificity of the resulting antibodies. These methods were found togenerate antibodies that bind to particular CC chemokines and some whichblock binding of vaccinia virus vCCI protein to CC chemokines(RANTES/CCL5).

Sequential immunization or a vertebrate, preferably a mouse, withdifferent chemokines proceeds by, for example, immunizing the animalwith a first chemokine in an appropriate adjuvant, boosting severalweeks later with a second chemokine, and finally boosting a second timewith a third chemokine. However, antikine antibodies may be produced bymethods involving multiple immunizations and boosts, for example, using2, 3, 4 or more different chemokines in different combinations. Bothprotein and DNA-based chemokine immunizations may be performed. DNAimmunization is well-known in the art and incorporated by reference toAntibodies: A Laboratory Manual (Eds. E. Harlow & D. Lane, 1988). Thepolynucleotide sequences of chemokines are well-known in the art andalso incorporated by reference to Yoshie, et al., Adv. Immunol. 78:57-110 (2001). Immunizations may be performed using adjuvants orconjugates of chemokines and carrier proteins such as those described byAntibodies: A Laboratory Manual, Eds. E. Harlow & D. Lane (1988), whichis incorporated by reference. Preferably, about 500 μg/kg body weight orabout 10 μg of a chemokine per mouse is administered and preferredadjuvants are Complete Freund's adjuvant for initial immunizations andIncomplete Freund's adjuvant for subsequent boosts. Exemplary CCchemokines for use as immunogens include at least two of CCL2/MCP-1,CCL3/MIP-1α, CCL4/MIP-1β and CCL5/RANTES.

In one embodiment of sequential immunization, a mouse is first immunizedwith MIP-1α and subsequently boosted with RANTES and/or MCP-1, or anyother sequence of the CC chemokines RANTES, MIP-1α, MIP-1β and/or MCP-1.Mouse serum is tested for reactivity with RANTES, MIP-1α, MIP-1β andMCP-1 by ELISA. A spleen from a mouse with appropriate serum responsesis then used to proceed with hybridoma production.

The antibodies produced by the hybridoma cell lines can be tested fortheir ability to recognize CC chemokines by ELISA and for blockingactivity in either in vitro chemotaxis assays or in vCCI/chemokinebinding assays. Using this strategy several unique monoclonal antibodieswhich bind and inhibit multiple CC chemokines were identified. Theseantibodies were designated “antikines”. The hybridoma-producedantibodies may also be tested in other functional assays. Hybridoma celllines producing the antibodies with multiple CC chemokine reactivitiesare then cloned and expanded for antibody production and purification.Screening assays to determine antibody binding specificity are wellknown and routinely practiced in the art. For a comprehensive discussionof such assays, see Harlow, et al. (Eds.), Antibodies: A LaboratoryManual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y., Chapter6 (1988). After its initial identification, an antikine antibody mayundergo affinity enhancement, for example, by a process of affinitymaturation or by engineering of its CDR or framework sequences; or itmay be humanized. For use in human therapy, preferably, an antikineantibody will be fully human or humanized and recognize two, three, fouror more CC chemokines.

In one embodiment, methods for the screening of antibodies that possessthe desired specificity include, but are not limited to, enzyme-linkedimmunosorbent assay (ELISA) and other immunologically-mediatedtechniques known within the art. In a specific embodiment, selection ofantibodies that are specific to a particular domain of CC-chemokines isfacilitated by generation of hybridomas that bind to the fragment ofCC-chemokines possessing such a domain. Antibodies that are specific forone or more domains within CC-chemokines, e.g., conserved domains ofCC-chemokine family proteins, or derivatives, fragments, analogs orhomologs thereof, are also provided herein.

The antikine antibodies and fragments thereof of the invention may beassayed for specific binding to the CC-chemokines, particularly MIP-1α,MIP-1β, RANTES, MCP-1 and/or other chemokines, in competitive andnon-competitive binding immunoassays. Well-known procedures forimmunoassays are hereby incorporated by reference to Stites and Terr(Eds.) Basic and Clinical Immunology, 7th ed., (1991); Maggio (Ed.)Enzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Tijan, Practiceand Theory of Enzyme Immunoassays, Laboratory Techniques in Biochemistryand Molecular Biology, Elsevier Science Publishers B.'V., Amsterdam(1985); Harlow and Lane (Eds.) Antibodies, a Laboratory Manual, ColdSpring Harbor, N.Y. (1988); Chan (Ed.), Immunoassay: A Practice Guide,Academic Press, Orlando, Fla. (1987); Price and Newman (Eds.) Principlesand Practice of Immunoassays, Stockton Press, N.Y. (1991); and Ngo (Ed.)(1988) Non-isotopic Immunoassays, Plenum Press, N.Y. (1988).

Immunoassays to measure antibody binding can be either competitive ornoncompetitive. In general in the antibody context, a competitive assayinvolves competition for binding of a ligand between two antibodies. Forexample, a labeled MCP-1 may be used to assess whether one antibody cancompete with another antibody for binding the labeled MCP-1. The assaymay be based on such standard assays as the enzyme linked immunosorbentassay (ELISA) or radioimmunoassay (RIA) for example.

Alternatively, the antibodies and antibody fragments of the inventionmay be tested in noncompetitive assays for binding to substrates. Forinstance, a standard ELISA may be used in which the ligand (e.g., MCP-1)is immobilized on an ELISA plate. A test antibody is incubated with theligand and allowed to bind. The plate is washed and thereafter, anenzyme-conjugated, secondary antibody (e.g., a mouse anti-human Fcantibody) binds to the test antibody if the test antibody is bound tothe ligand. After washing, a substrate for the enzyme is added andallowed to react with the enzyme. Generally a color change indicates thepresence of an antibody that reacts with the ligand. The ELISA may berepeated for different CC-chemokines to determine which chemokines arerecognized by the test antibody.

Immunoassays often use labeled assay components. The label can be in avariety of forms and may be coupled directly or indirectly to thedesired component of the assay according to methods well known in theart. Common labels for assay components include radioactive isotopes,including ³H, ¹²⁵I, ³⁵S, ¹⁴C, and ³²P, fluorophores, chemiluminescentagents, and enzymes. The choice of a particular label will depend on thesensitivity required, the ease of conjugation with the compound, thestability requirements, and the available instrumentation, and will beeasily determined by one of ordinary skill in the art.

Assays to assess whether the antibodies of the invention inhibitCC-chemokine activity, particularly MIP-1α, MIP-1f3, RANTES, MCP-1and/or other chemokines, may be easily performed using known assays forchemotaxis, intracellular calcium increase, and the like. For example,but not by way of limitation, chemokine chemotaxis assays may beperformed in 96 well plastic chambers. The wells are separated by afilter into two compartments. The filter allows the passage of cellsfrom one compartment to the next in response to chemical gradients. Testcells are placed in one compartment of the chamber in a culture mediumand a CC-chemokine, for example, is placed in culture medium in theother compartment. Cells traversing the filter are counted. In otherwells, the CC-chemokine is mixed with the test antibody to determine ifthe antibody is able to block cell migration.

For further antibody engineering of the antikine monoclonals identifiedby the screening procedures above, the DNA sequences of the antibodieswere determined as reported in Example 7.

The affinity of the antikine antibodies of the invention may be furtherimproved using methods known in the art, such as those described byRaipal, et al., Proc. Natl. Acad. Sci. USA. 102:8466-8471 (2005);Lippow, et al. Nat. Biotechnol. 25:1171-1176 (2007); Wu, et al., J. Mol.Biol. 368: 652-665 (2007); Yang, et al., J. Mol. Biol. 254: 392-403(1995); and Huse, et al., J. Immunol. 149:3903-3913 (1992) which areeach incorporated by reference as teaching methods for affinitymaturation or enhancement of antibodies. The antikine antibodies of theinvention can have affinities of less than 200, 100 nM (e.g., theoriginally isolated murine antibody 3C12F has an affinity of 49 nM forMIP-1α) and can be enhanced to be less than 40, 30, 20, 10, 5, 1, 0.5 or0.1 nM during affinity maturation procedures. Procedures useful for theaffinity maturation of monoclonal antibodies are well known in the artand are incorporated by reference to Antibody Engineering (Humana Press,2004) and Phage Display, T. Clackson and H. B. Lowman, editors (OxfordUniversity Press, 2004). Such procedures can start with the chimericform of such an antikine antibody, which can be humanized eitherseparately or concurrently with the affinity maturation. The variableregions of the antibody may be expressed as a Fab fragment on thesurface of filamentous phage. A mutagenesis strategy may be employed tochange each residue within the six CDRs to make a combinatorial libraryof Fab fragments expressed on phage. These can be screened for bindingto chemokine antigens and analyzed for improvements in affinity.Subsequently preferred amino acid substitutions can be combined for evengreater improvements in affinity. At the same time mutations within theframework regions of the variable domains can be analyzed to findsequences with improved properties.

A “variant” or “analog” antibody, as described above differs in aminoacid sequence from a parent antibody amino acid sequence by virtue ofaddition, deletion and/or substitution of one or more amino acidresidue(s) in the parent antibody sequence. In one embodiment, thevariant comprises one or more amino acid substitution(s) in one or morehypervariable region(s) of the parent antibody.

An antibody analog may be engineered by replacing 1, 2, 3, 4, 5, 6, 7, 8or more amino acid residues in a CDR, hypervariable or framework regionof a heavy or light chain sequence of 3C12F, 7D1G, 7D12A, 18V4F, or18P7E. Similarly, the isotypes of such an antibody may be changed byisotype switching techniques known in the art or by genetic engineeringprocedures, such as CDR grafting, chimeric antibody formation, orhumanization.

Analogs may be produced by a process of affinity maturation of 3C12F,7D1G, 7D12A, 18V4F or 18P7E type monoclonal antibodies. Analogsgenerally selected for a higher binding affinity or a broader chemokinebinding profile which may be acquired at the same time. Analogs may beeasily identified by determining whether they bind to the samechemokines to which the parent antibodies bind, for example, by ELISA orother well-known assays. An analog includes those variants in which theheavy and light chains share about 90, 95, 99 or 100% sequence identitywith the corresponding heavy and light chain sequences of 3C12F, 7D1G,7D12A, 18V4F, or 18P7E. Affinity matured variants of antibodies producedby 3C12F, 7D1G, 7D12A, 18V4F or 18P7E hybridoma cell lines orsubcultures thereof can be selected to have binding affinities of 1,000,800, 400, 200, 100, 75, 50, 40, 30, 20, 10, 5, 1 or 0.1 nM or less. Someanalogs or variants will have one, two, three, four, five, six, seven,ten, or up to twenty amino acid sequence modifications, such asdeletions, insertions or substitutions, an analog can have about 2 to 10amino acid substitutions in one or more hypervariable regions or CDRs ofthe 3C12F, 7D1G, 7D12A, 18V4F or 18P7E antibodies.

An analog may constitute an antibody, or antigen-binding fragmentthereof,which binds two or more of MIP-1α, MIP-1β, RANTES, MCP-1 which comprisesat least one of the following CDR combinations: CDR1 and CDR2; CDR1 andCDR3; CDR2 and CDR3; and CDR1, CDR2, and CDR3, of the 3C12F, 7D1G,7D12A, 18V4F or 18P7E heavy chain and/or light chain variable regions.

Chimeric antibodies in which an animal antigen-binding variable domainis coupled to a human constant domain are well known in the art andmethods for making them are incorporated by reference to Cabilly et al.,U.S. Pat. No. 4,816,567; Morrison, S. L. et al., Proc. Natl. Acad. Sci.USA 81:6851-6855 (1984); Boulianne, G. L. et al., Nature 312:643-646(1984); and Neuberger, M. S. et al., Nature 314:268-270 (1985).

The isotype of the human constant domain may be selected to tailor thechimeric antibody for participation in antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity (see e.g.,Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987); Riechmann, L.et al., Nature 332:323-327 (1988); Love et al., Methods in Enzymology178:515-527 (1989); Bindon, et al., J. Exp. Med. 168:127-142 (1988),which are incorporated by reference. Chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT InternationalApplication No. PCT/US86/02269; European Patent Application No. 184,187;European Patent Application No. 171,496; European Patent Application No.173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No.4,816,567; European Patent Application No. 125,023; Better et al.,Science 240:1041-1043 (1988).

The antikine antibodies of the invention may be humanized by knownprocedures including those disclosed by Carter, U.S. Pat. No. 6,719,971,Almagro, et al., Front. Biosci. 13:1619-1633 (2008); Pini, et al., Comb.Chem. High Throughput Screen. 5:503-510 (2002); and Wu, et al., J. Mol.Biol. 294:151-162 (1999) which are incorporated by reference.Humanization produces an immunoglobulin molecule with the antibodyspecificity of the donor animal (murine) antibody, but with reducedimmunogenicity and better effector functions in humans. This processinvolves embedding known antikine CDR sequences into the light and heavychain variable region framework sequences of a human antibody. Ingeneral, humanized antibodies are produced by substituting mouse CDRsinto a human variable domain framework which is most likely to result inretention of the correct spatial orientation of the CDRs if the humanvariable domain framework adopts the same or similar conformation to themouse variable framework from which the CDRs originated. This isachieved by obtaining the human variable domains from human antibodieswhose framework sequences exhibit a high degree of sequence identitywith the murine variable framework domains from which the CDRs werederived. The heavy and light chain variable framework regions can bederived from the same or different human antibody sequences. The humanantibody sequences can be the sequences of naturally occurring humanantibodies or can be consensus sequences of several human antibodies.See Kettleborough, et al., Protein Engineering 4:773-783 (1991);Kolbinger, et al., Protein Engineering 6:971-980 (1993) and Carter, etal., WO 92/22653.

Having identified the complementarity determining regions of the murinedonor immunoglobulin and appropriate human acceptor immunoglobulins, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized antibody. In general, substitution of human amino acidresidues with murine should be minimized, because introduction of murineresidues increases the risk of the antibody eliciting ahuman-anti-mouse-antibody (HAMA) response in humans.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

The selection of amino acid residues for substitution is determined, inpart, by computer modeling. In general, molecular models are producedstarting from solved structures for immunoglobulin chains or domainsthereof. The chains to be modeled are compared for amino acid sequencesimilarity with chains or domains of solved three-dimensionalstructures, and the chains or domains showing the greatest sequencesimilarity is/are selected as starting points for construction of themolecular model. Chains or domains sharing at least 50% sequenceidentity are selected for modeling, and preferably those sharing atleast 60%, 70%, 80%, 90%, 95% sequence identity or more are selected formodeling. The solved starting structures are modified to allow fordifferences between the actual amino acids in the immunoglobulin chainsor domains being modeled, and those in the starting structure. Themodified structures are then assembled into a composite immunoglobulin.Finally, the model is refined by energy minimization and by verifyingthat all atoms are within appropriate distances from one another andthat bond lengths and angles are within chemically acceptable limits.

The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a murine variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid should usually be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid: noncovalently binds antigendirectly, is adjacent to a CDR region, otherwise interacts with a CDRregion (e.g., is within about 3-6 Å of a CDR region as determined bycomputer modeling), or participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, van derWaals forces, hydrophobic interactions, and the like.

Residues which are “adjacent to a CDR region” include amino acidresidues in positions immediately adjacent to one or more of the CDRs inthe primary sequence of the humanized immunoglobulin chain, for example,in positions immediately adjacent to a CDR as defined by Kalbat—Wu &Kabat, J. Exp. Med. 132:211-250 (1970)—or a CDR as defined byChothia—Chothia & Lesk J. Mol. Biol. 196:901-917 (1987). These aminoacids are particularly likely to interact with the amino acids in theCDRs and, if chosen from the acceptor, to distort the donor CDRs andreduce affinity. Moreover, the adjacent amino acids may interactdirectly with the antigen, Amit, et al., Science 233:747-753 (1986),which is incorporated herein by reference, and selecting these aminoacids from the donor may be desirable to keep all the antigen contactsthat provide affinity in the original antibody.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to affect a CDR region. Residues that “otherwiseinteract with a CDR region” may be identified by analyzing athree-dimensional model of the donor immunoglobulin (e.g., acomputer-generated model). A three-dimensional model, typically of theoriginal donor antibody, shows that certain amino acids outside of theCDRs are close to the CDRs and have a good probability of interactingwith amino acids in the CDRs by hydrogen bonding, van der Waals forces,hydrophobic interactions, etc. At those amino acid positions, the donorimmunoglobulin amino acid rather than the acceptor immunoglobulin aminoacid may be selected. Amino acids according to this criterion willgenerally have a side chain atom within about 3 Å of some atom in theCDRs and must contain an atom that could interact with the CDR atomsaccording to established chemical forces, such as those listed above.

Amino acids that are capable of interacting with amino acids in the CDRsmay be identified in yet another way. The solvent accessible surfacearea of each framework amino acid is calculated in two ways: (1) in theintact antibody, and (2) in a hypothetical molecule consisting of theantibody with its CDRs removed. A significant difference between thesenumbers of about 10 Å² or more shows that access of the framework aminoacid to solvent is at least partly blocked by the CDRs, and thereforethat the amino acid is making contact with the CDRs. Solvent accessiblesurface area of an amino acid may be calculated based on athree-dimensional model of an antibody, using algorithms known in theart; e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee & Richards,J. Mol. Biol. 55:379 (1971), both of which are incorporated herein byreference. Framework amino acids may also occasionally interact with theCDRs indirectly, by affecting the conformation of another frameworkamino acid that in turn contacts the CDRs.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA82:4592-66 (1985) or Chothia & Lesk, supra. Generally, unusual packingresidues should be retained in the humanized antibody if they differfrom those in the human frameworks.

In general, one or more of the amino acids fulfilling the above criteriais substituted. In some embodiments, all or most of the amino acidsfulfilling the above criteria are substituted. Occasionally, there issome ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant immunoglobulins so produced can be tested in any ofthe assays described herein for the desired activity, and the preferredimmunoglobulin selected.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor antibody. Although not usually desirable, it is sometimespossible to make one or more conservative amino acid substitutions ofCDR residues without appreciably affecting the binding affinity of theresulting humanized immunoglobulin. By conservative substitutions isintended combinations such as gly, ala; val, ile, leu; asp, glu; asn,gln; ser, thr; lys, arg; and phe, tyr.

Additional candidates for substitution are acceptor human frameworkamino acids that are unusual or “rare” for a human immunoglobulin atthat position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor antibody or from theequivalent positions of more typical human immunoglobulins. For example,substitution may be desirable when the amino acid in a human frameworkregion of the acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is common for thatposition in human immunoglobulin sequences; or when the amino acid inthe acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is also rare,relative to other human sequences. These criteria help ensure that anatypical amino acid in the human framework does not disrupt the antibodystructure. Moreover, by replacing an unusual human acceptor amino acidwith an amino acid from the donor antibody that happens to be typicalfor human antibodies, the humanized antibody may be made lessimmunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20% but usually less than about 10% ofsequences in a representative sample of sequences, and the term“common”, as used herein, indicates an amino acid occurring in more thanabout 25% but usually more than about 50% of sequences in arepresentative sample. For example, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Wu & Kabat, supra). Whendeciding whether an amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences in the same subgroup as the acceptorsequence. Additional candidates for substitution are acceptor humanframework amino acids that would be identified as part of a CDR regionunder the alternative definition proposed by Chothia & Lesk, supra.

Other candidates for substitution are acceptor framework residues thatcorrespond to a rare or unusual donor framework residue. Rare or unusualdonor framework residues are those that are rare or unusual (as definedherein) for murine antibodies at that position. For murine antibodies,the subgroup can be determined according to Kabat and residue positionsidentified which differ from the consensus. These donor specificdifferences may point to somatic mutations in the murine sequence thatenhances activity. Unusual residues that are predicted to affect bindingare retained, whereas residues predicted to be unimportant for bindingcan be substituted.

More candidates for substitution are non-germline residues occurring inan acceptor framework region. For example, when an acceptor antibodychain (i.e., a human antibody chain sharing significant sequenceidentity with the donor antibody chain) is aligned to a germlineantibody chain (likewise sharing significant sequence identity with thedonor chain), residues not matching between acceptor chain framework andthe germline chain framework can be substituted with correspondingresidues from the germline sequence.

Other than the specific amino acid substitutions discussed above, theframework regions of humanized immunoglobulins are usually substantiallyidentical, and more usually, identical to the framework regions of thehuman antibodies from which they were derived. Of course, many of theamino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. Thus, in one embodiment thevariable framework region of the humanized immunoglobulin shares atleast 85% sequence identity to a human variable framework regionsequence or consensus of such sequences. In another embodiment, thevariable framework region of the humanized immunoglobulin shares atleast 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequenceidentity to a human variable framework region sequence or consensus ofsuch sequences. In general, however, such substitutions are undesirable.

In some embodiments, humanized antibodies preferably exhibit a specificbinding affinity for antigen similar to or higher than that of the mouseantibody from which they were constructed. Usually the upper limit ofbinding affinity of the humanized antibodies for antigen is within afactor of three, four or five of that of the donor immunoglobulin. Oftenthe lower limit of binding affinity is also within a factor of three,four or five of that of donor immunoglobulin. Alternatively, the bindingaffinity can be compared to that of a humanized antibody having nosubstitutions (e.g., an antibody having donor CDRs and acceptorframework regions, but no framework region substitutions). In suchinstances, the binding of the antibody (with substitutions) ispreferably at least two- to three-fold greater, or three- to four-foldgreater, than that of the unsubstituted antibody. For makingcomparisons, activity of the various antibodies can be determined, forexample, by BIAcore® (i.e., surface plasmon resonance using unlabelledreagents) or competitive binding assays.

An engineered antibody may be designed based on the sequences of themonoclonal antibodies disclosed herein using standard affinitymaturation techniques, such as those described Wu, et al., J. Mol. Biol.350: 126-144 (2005) (incorporated by reference) in order to increase theaffinity of the antibody to the originally identified chemokines and/orbroaden the chemokine selectivity to other CC-chemokines which may alsobe involved in inflammatory diseases.

The class or isotype or subclass (e.g., IgG1, IgG2, IgG3 or IgG4) may beselected, particular for chimeric, humanized or engineered antibodyproducts to target or adapt the antibody to a particular function wellknown for the selected class or subclass. For example, human IgG2 can beselected to minimize passage of the antibody product across the placentaor minimize Fc-receptor interaction with other components a subject'simmune system, and subclass IgG4 to minimize the ability of the antibodyproduct to activate complement. IgA isotype can be selected to produce asecretory antibody and a pentameric IgM antibody product to enhancebinding of the antibody compared to monomeric antibodies. The functionsof various classes and subclasses of antibody molecules are incorporatedby reference to Kuby, Immunology, WH Freeman (1997).

Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of an antikine antibody, wherein these domains are presentin a single polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding; seePlückthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)which is incorporated by reference. Single-chain antibodies specific toCC chemokines may be produced by known methods such as those disclosedby U.S. Pat. No. 4,946,778 which is incorporated by reference.

“Diabodies” refers to small antibody fragments with two antigen-bindingsites, which fragments comprise a heavy chain variable domain (V_(H))connected to a light chain variable domain (V_(L)) in the samepolypeptide chain (V_(H) and V_(L)). By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites. The antikine antibodies of theinvention may be formulated as diabodies which can be made by proceduresincorporated by reference to EP 404,097; WO 93/11161; and Hollinger etal., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

“Linear antibodies” refers to the antibodies described in Zapata et al.Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprisea pair of tandem Fd segments (V_(H)-C_(H) 1-V_(H)-C_(H) 1) which form apair of antigen binding regions. Linear antibodies can be bispecific ormonospecific. The antikine antibodies of the invention may be producedaccording to the procedures described by Zapata, et al., which arehereby incorporated by reference.

Fab expression libraries may be constructed by other conventionalmethods such as those disclosed by and incorporated by reference to Huseet al., Science 246:1275-1281 (1989) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor CC chemokines or derivatives, fragments, analogs or homologsthereof.

Unique portions of the antibodies exemplified below, such as their CDRsequences and sequences containing a portion of a CDR, may be used toproduce anti-idiotype antibodies. These antibodies recognize one or moreidiotypes on a variable segment of an antikine antibody and can be usedto identify or purify an antikine antibody or modulate its activity.Such anti-idiotype antibodies can be produced by procedures known in theart, such as by conjugation of a variable peptide sequence of anantibody to an immunogenic carrier and repeated immunization. Suchmethods are also incorporated by reference to Cavenaugh, et al., Pharm.Res. 21:1480-1488 (2004).

An anti-idiotypic antibody is an antibody that recognizes determinantsof another antibody (a target antibody). Generally, the anti-idiotypicantibody recognizes determinants of the antigen-binding site of thetarget antibody. Typically, the target antibody is a monoclonalantibody. An anti-idiotypic antibody is generally prepared by immunizingan animal (particularly, mice) of the same species and genetic type asthe source of the target monoclonal antibody, with the target monoclonalantibody. The immunized animal mounts an immune response to theidiotypic determinants of the target monoclonal antibody and producesantibodies against the idiotypic determinants of the target monoclonalantibody. Antibody-producing cells, such as splenic cells, of theimmunized animal may be used to generate anti-idiotypic monoclonalantibodies. Furthermore, an anti-idiotypic antibody may also be used toimmunize animals to produce anti-anti-idiotypic antibodies. Theseimmunized animals may be used to generate anti-anti-idiotypic monoclonalantibodies using standard techniques. The anti-anti-idiotypic antibodiesmay bind to the same epitope as the original, target monoclonal antibodyused to prepare the anti-idiotypic antibody. The anti-anti-idiotypicantibodies represent other monoclonal antibodies with the same antigenspecificity as the original target monoclonal antibody.

If the binding of the anti-idiotypic antibody with the target antibodyis inhibited by the relevant antigen of the target antibody, and if theanti-idiotypic antibody induces an antibody response with the samespecificity as the target antibody, it mimics the antigen of the targetantibody. Such an anti-idiotypic antibody is an “internal imageanti-idiotype” and is capable of inducing an antibody response as if itwere the original antigen, see Bona and Kohler, Anti-idiotypicAntibodies and Internal Image in Monoclonal and Anti-idiotypicAntibodies: Probes for Receptor Structure and Function, Venter J. C.,Frasser, C. M., Lindstrom, J. (Eds.), Alan R. Liss, N.Y., pp 141-149(1984). Vaccines incorporating internal image anti-idiotype antibodieshave been shown to induce protective responses against viruses,bacteria, and parasites; Kennedy, et al. Science 232:220-223 (1986);McNamara, et al., Science 226:1325-1326 (1985). Internal imageanti-idiotypic antibodies have also been shown to induce immunity totumor related antigens; Raychauhuri, et al., J. Immunol. 137:1743-1749(1986); Raychauhuri et al., J. Immunol. 139:3902-3910 (1987);Bhattacharya-Chatterjee et al., J. Immunol. 139:1354-1360 (1987);Bhattacharya-Chatterjee, et al., J. Immunol. 141:1398-1403 (1988). Theteachings of the documents cited in this paragraph are incorporated byreference.

Anti-idiotypic antibodies for CC chemokines may be prepared, forexample, by immunizing an animal, such as a mouse, with a immunogenicamount of a composition comprising CC-chemokines or immunogenic portionsthereof, containing at least one antigenic epitope of CC-chemokines. Thecomposition may also contain a suitable adjuvant, and any carriernecessary to provide immunogenicity. Monoclonal antibodies recognizingCC-chemokines may be prepared from the cells of the immunized animal asdescribed above. A monoclonal antibody recognizing a common epitope ofCC chemokines is then selected and used to prepare a compositioncomprising an immunogenic amount of the anti-CC-chemokine monoclonalantibody. Typically, a 25 to 200 μg dose of purified CC-chemokinemonoclonal would be sufficient in a suitable adjuvant. Animals may beimmunized 2-6 times at 14 to 30 day intervals between doses. Typically,animals are immunized by any suitable route of administration, such asintraperitoneal, subcutaneous, intravenous or a combination of these.Anti-idiotypic antibody production may be monitored during theimmunization period using standard immunoassay methods. Animals withsuitable titers of antibodies reactive with the target monoclonalantibodies may be re-immunized with the monoclonal antibody used as theimmunogen three days before harvesting the antibody producing cells.Preferably, spleen cells are used, although other antibody producingcells may be selected. Antibody-producing cells are harvested and fusedwith myeloma cells to produce hybridomas, as described above, andsuitable anti-idiotypic antibody-producing cells are selected.

Anti-anti-idiotypic antibodies are produced by another round ofimmunization and hybridoma production by using the anti-idiotypicmonoclonal antibody as the immunogen.

As described above, the variable segments of antikine antibodies, suchas CDR segments, or peptide fragments of variable segments, includingfragments having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more contiguousamino acid residues, especially those that have binding activity forchemokines, may be used for a number of applications in addition totherapeutic treatment of autoimmune diseases, including as peptide-basedpharmaceutical agents, vaccines, production of anti-idiotype antibodies,or as immunogens. Another aspect of the present invention is directed tomethods of inducing an immune response in a mammal against a polypeptideof the invention by administering to the mammal an amount of thepolypeptide preparation sufficient to induce an immune response. Theamount will be dependent on the animal species, size of the animal, andthe like but can be determined by those skilled in the art.

The invention also includes antibodies or antibody fragments derivedfrom chimeric or humanized antikine antibodies through the process ofaffinity maturation. Amino acid substitutions within the CDRs may beidentified which significantly improve the affinity of the antibody forCC-chemokines and are therefore included herein. Methods for affinitymaturation are described for example in Wu et al., Proc. Natl. Acad.Sci. USA 95:6037-6042 (1998) and by Clackson and Loman, Phage Display,Oxford University Press (2004) each of which is also incorporated byreference.

Anti-CC chemokine antikine antibodies may be used in methods knownwithin the art relating to the localization and/or quantification of aCC-chemokines (e.g., for use in measuring levels of CC-chemokines withinappropriate physiological samples, for use in diagnostic methods, foruse in imaging the protein, and the like). In a given embodiment,antibodies for CC-chemokines, or derivatives, fragments, analogs orhomologs thereof, that contain the antibody derived binding domain, areutilized as pharmacologically-active compounds, drugs or therapeuticcompounds.

An anti-CC-chemokine antikine antibody (e.g., monoclonal antibody) canbe used to isolate CC-chemokines by standard techniques, such asaffinity chromatography or immunoprecipitation. An anti-CC-chemokineantibody can facilitate the purification of natural CC-chemokines fromcells and of recombinantly produced CC-chemokines expressed in hostcells. Moreover, an anti-CC-chemokine pan-antibody can be used to detectCC-chemokines (e.g., in a cellular lysate or cell supernatant) in orderto evaluate the abundance and pattern of expression of theCC-chemokines. Anti-CC-chemokine antikine antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure in order to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, and ³H.

In addition, the antibodies of the present invention may be conjugatedto toxins such as radioisotopes, protein toxins and chemical toxinswhich may be conjugated to antibodies. Such toxins include, but are notlimited to Lead-212, Bismuth-212, Astatine-211, Iodine-131, Scandium-47,Rhenium-186, Rhenium-188, Yttrium-90, Iodine-123, Iodine-125,Bromine-77, Indium-111, Boron-10, Actinide, ricin, adriamycin,calicheamicins, 5-fluorouracil, auristatins, and maytansinoids.

Chimeric, humanized, and human antibodies as well as antigen-bindingfragments thereof are typically produced by recombinant expression.Nucleic acids encoding humanized light and heavy chain variable regions,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding immunoglobulinchains are operably linked to control sequences in the expressionvector(s) that ensure the expression of immunoglobulin polypeptides.Expression control sequences include, but are not limited to, promoters(e.g., naturally-associated or heterologous promoters), signalsequences, enhancer elements, and transcription termination sequences.Preferably, the expression control sequences are eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and the collection andpurification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences, see, e.g., Itakura, et al., U.S. Pat. No.4,704,362, which is incorporated by reference. E. coli is oneprokaryotic host particularly useful for cloning the polynucleotides(e.g., DNA sequences) of the present invention. Other microbial hostssuitable for use include bacilli, such as Bacillus subtilus, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(e.g., polynucleotides encoding immunoglobulins or fragments thereof).See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting heterologous proteins (e.g., intactimmunoglobulins) have been developed in the art, and include CHO celllines, various Cos cell lines, HeLa cells, preferably, myeloma celllines, or transformed B-cells or hybridomas. Preferably, the cells arenonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, and anenhancer, Queen et al., Immunol. Rev. 89:49-68 (1986), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus,cytomegalovirus and the like. See Co, et al., J. Immunol. 148:1149-1154(1982), each of the above documents is incorporated by reference.

Alternatively, antibody-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal, as describedfor example by Deboer, et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat.No. 5,304,489, and Meade, et al., U.S. Pat. No. 5,849,992, each of whichis incorporated by reference. Suitable transgenes include codingsequences for light and/or heavy chains in operable linkage with apromoter and enhancer from a mammary gland specific gene, such as caseinor beta lactoglobulin.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences or fragments thereof andexpression control sequences) can be transferred into the host cell bywell-known methods, which vary depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics or viral-based transfection may be used forother cellular hosts. Other methods used to transform mammalian cellsinclude the use of polybrene, protoplast fusion, liposomes,electroporation, and microinjection (see generally, Sambrook, et al.,supra). For production of transgenic animals, transgenes can bemicroinjected into fertilized oocytes, or can be incorporated into thegenome of embryonic stem cells, and the nuclei of such cells transferredinto enucleated oocytes. All of these methods are incorporated byreference to Sambrook, et al., Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Press, 2nd ed. (1989).

When heavy and light chains are cloned on separate expression vectors,the vectors are co-transfected to obtain expression and assembly ofintact immunoglobulins. Once expressed, the whole antibodies, theirdimers, individual light and heavy chains, or other immunoglobulin formsmay be purified according to standard procedures of the art, includingby use of ammonium sulfate precipitation, affinity columns, columnchromatography, HPLC purification, gel electrophoresis and other similarprocedures including those disclosed by Scopes, Protein Purification,Springer-Verlag, N.Y. (1982) which is incorporated by reference. Forpharmaceutical use, it is desirable to employ substantially pureimmunoglobulins having a degree or purity and/or homegeneity of at least90 to 95%, or even 98 to 99%.

Fragments of, or truncated, antikine antibodies, such as Fab fragment,Fab′ fragment, a F(ab′)₂ fragment, and F_(v) fragment, as well as singleantibody chains comprising individual light and/or heavy chain CDRsinvolved in chemokine binding may be used to bind to or neutralize oneor more CC chemokines.

Antikine antibodies may be chemically modified or derivatized to providea desired effect.

An antibody conjugate or conjugated antibody may be made using antikineantibody or its fragment joined via peptide bonds to a heterologousprotein. In such embodiments, the antigen-binding portion of theantibody binds MIP-1α, MIP-1β, RANTES, MCP-1, and/or other relatedCC-chemokines. Antikine antibodies may also be conjugated to enzymes,toxins or a cytokine as effector moieties. They may also contain or befurther conjugated or covalently linked to accessory moieties such aschemical or radiological tags, toxins, biologically active or targetingmoieties, or other substances which increase their biological half livesor biological availability, such as by conjugation to polyethyleneglycol or albumin. An antikine antibody of the invention may contain orbe conjugated to an accessory moiety, such as those disclosed by Carterand Senter, Cancer J. 14: 154-169 (2008) which is incorporated byreference. The antibodies and antibody products of the invention can beimmobilized to a solid substrate or immunoaffinity resin such as thosedescribed by Antibodies: A Laboratory Manual; Eds. E. Harlow & D. Lane,(1988) which is incorporated by reference.

Pegylation of antibodies and antibody fragments of the invention may becarried out by any of the pegylation reactions known in the art, asdescribed, for example, in the following references: Focus on GrowthFactors 3:4-10 (1992); EP 0 154 316; and EP 0 401 384, each of which isincorporated by reference herein in its entirety. Preferably, thepegylation is carried out via an acylation reaction or an alkylationreaction with a reactive polyethylene glycol (PEG) molecule (or ananalogous reactive water-soluble polymer). A preferred water-solublepolymer for pegylation of the antibodies and antibody fragments of theinvention is PEG. As used herein, “polyethylene glycol” is meant toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono (Cl—ClO) alkoxy- or aryloxy-polyethyleneglycol.

Methods for preparing pegylated antibodies and antibody fragments of theinvention will generally comprise the steps of (a) reacting the antibodyor antibody fragment with PEG, such as a reactive ester or aldehydederivative of PEG, under conditions whereby the antibody or antibodyfragment becomes attached to one or more PEG groups, and (b) obtainingthe reaction products. It will be apparent to one of ordinary skill inthe art to select the optimal reaction conditions or the acylationreactions based on known parameters and the desired result.

Pegylated antibodies and antibody fragments may generally be used totreat conditions that may be alleviated or modulated by administrationof the antibodies and antibody fragments described herein. Generally thepegylated antibodies and antibody fragments have increased half-life, ascompared to the nonpegylated antibodies and antibody fragments. Thepegylated antibodies and antibody fragments may be employed alone,together, or in combination with other pharmaceutical compositions.

In other embodiments of the invention the antibodies or antigen-bindingfragments thereof are conjugated to albumin using techniques recognizedin the art.

In another embodiment of the invention, antibodies, or fragmentsthereof, are modified to reduce or eliminate potential glycosylationsites. Such modified antibodies are often referred to as “aglycosylated”antibodies. In order to improve the binding affinity of an antibody orantigen-binding fragment thereof, glycosylation sites of the antibodycan be altered, for example, by mutagenesis (e.g., site-directedmutagenesis). “Glycosylation sites” refer to amino acid residues whichare recognized by a eukaryotic cell as locations for the attachment ofsugar residues. The amino acids where carbohydrate, such asoligosaccharide, is attached are typically asparagine (N-linkage),serine (O-linkage), and threonine (O-linkage) residues. In order toidentify potential glycosylation sites within an antibody orantigen-binding fragment, the sequence of the antibody is examined, forexample, by using publicly available databases such as the websiteprovided by the Center for Biological Sequence Analysis (seehttp://www.cbs.dtu.dk/services/NetNGlyc/ for predicting N-linkedglycoslyation sites and http://www.cbs.dtu.dk/services/NetOGlyc/ forpredicting O-linked glycoslyation sites). Additional methods foraltering glycosylation sites of antibodies are described in U.S. Pat.Nos. 6,350,861 and 5,714,350 which are incorporated by reference.

In yet another embodiment of the invention, antibodies or fragmentsthereof can be altered by modifying the constant region of the antibodyto reduce at least one constant region-mediated biological effectorfunction relative to an unmodified antibody. To modify an antibody ofthe invention such that it exhibits reduced binding to the Fc receptor,the immunoglobulin constant region segment of the antibody can bemutated at particular regions necessary for Fc receptor (FcR)interactions, see e.g., Canfield, S. M. and S. L. Morrison, J. Exp. Med.173:1483-1491 (1991); and Lund, J. et al., J. Immunol. 147:2657-266(1991) all of which are incorporated by reference. Reduction in FcRbinding ability of the antibody may also reduce other effector functionswhich rely on FcR interactions, such as opsonization and phagocytosisand antigen-dependent cellular cytotoxicity. Constant region derivativesto enhance or reduce antibody effector functions and to reduce or extendserum persistence are described by Carter, P. J., Nat. Rev. Immunol.6:343-357 (2006) which is incorporated by reference.

The antikine antibodies of the invention may be incorporated intopharmaceutical compositions such as those described above or incompositions used to store, preserve or administer the antibodies tosubjects in need of treatment.

Kits for use in the therapeutic or diagnostic applications above, maycontain one more antikine antibodies, positive or negative controlantibodies, CC chemokines bound by antikine antibodies, controlchemokines not bound by the antikine antibody, as well as otherpharmacological or diagnostic components and written instructionsregarding the use of the kit.

Antibodies or antigen-binding fragments of the invention are useful for,e.g., therapeutic purposes (by modulating activity of CC-chemokines),diagnostic purposes to detect or quantify CC-chemokines, andpurification of CC-chemokines. Therefore, kits comprising an antibody ofthe invention for any of the purposes described herein are also withinthe scope of the invention.

The CC chemokines, particularly MIP-1α, MIP-1β, RANTES, and/or MCP-1have been shown to play a role in pathological conditions associatedwith inflammation. MIP-1α, MIP-1β, RANTES, and/or MCP-1 have all beenshown to have potent chemotactic activity for leukocytes, especiallymonocytes and T lymphocytes. These pathological conditions include thosedescribed by Johnson et al., Trends Immunol. 26:268-274 (2005) which ishereby incorporated by reference.

MIP-1α, MIP-1β, RANTES, and/or MCP-1 are molecules with potentchemotactic activity for monocytes and T lymphocytes. Given theiroverlapping activities and the increased expression of all four of thesechemokines in human disease, blockade of two, three or four CC chemokinemolecules is expected to have a greater beneficial effect than justinhibition of a single CC chemokine alone. Accordingly, the antibodiesand antibody fragments of the invention are useful to modulate theactivity of these chemokines and affect the pathology of disordersassociated with these chemokines. As such, these antibodies andfragments are useful in therapeutic compositions for the treatment ofinflammatory conditions and pathological conditions associated withexpression of CC-chemokine molecules. In these embodiments, a patient isidentified as having one of the diseases to be treated, such as byexhibiting at least one sign or symptom of the disease or disorder. Atleast one antibody or antigen-binding fragment thereof of the inventionor compositions comprising at least one antibody or antigen-bindingfragment thereof of the invention is administered in a sufficient amountto alleviate at least one symptom of the disease or disorder, or toreduce the activity of at least one of MIP-1α, MIP-1β, RANTES, and/orMCP-1.

Disorders Amenable to Prevention or Treatment. As used herein, the terms“a disorder in which CC chemokine activity is detrimental” and “aCC-chemokine-associated disorder” are intended to include diseases andother disorders in which the presence of a CC-chemokine, includingMIP-1α, MIP-1β, RANTES, MCP-1 and other CC-chemokines, in a subjectsuffering from the disorder has been shown to be or is suspected ofbeing either responsible for the pathophysiology of the disorder or afactor that contributes to the disorder. Accordingly, a disorder inwhich CC-chemokine activity is detrimental is a disorder in whichinhibition of CC-chemokine activity is expected to prevent or alleviatethe symptoms and/or progression of the disorder. Such disorders may beevidenced, for example, by an increase in the concentration ofCC-chemokines in a biological fluid of a subject suffering from thedisorder, e.g., an increase in the concentration of RANTES in serum,plasma, synovial fluid, urine, etc. of the subject, which can bedetected, for example, using an anti-RANTES antibody. There are numerousexamples of disorders in which CC-chemokine activity is detrimental. Theuse of the antibodies and antibody portions of the invention in theprevention or treatment of specific disorders is discussed furtherbelow.

Rheumatoid arthritis (RA) is characterized by an influx of leukocytes,including T and B lymphocytes, macrophages, and neutrophils, into thesynovial lining of joints; see the review by Koch, Arthritis Rheum. 52:710-721 (2005). MIP-1α is found in high levels in synovial fluid of RApatients and the increased levels correlate with severity of disease.Similarly in rodent murine models of RA high levels of murine MIP-1α arefound in arthritic joints. Neutralizing antibodies decrease arthriticscores in a collagen-induced arthritis model by approximately 50%;Kasama, et al., J. Clin. Invest. 95: 2868-2876 (1995).

The levels of RANTES are also increased in joints in a rodentadjuvant-induced model of arthritis. Antibodies to RANTES reducedsymptoms in this model; Barnes et al., J. Clin. Invest. 101: 2910-2919(1998).

MCP-1 has also been shown to be produced by synovial cells andinfiltrating leukocytes in RA patients. Neutralizing antibodies to MCP-1also reduced the clinical score in a rodent model of arthritis; Ogata,et al., J. Pathol. 182: 106-114 (1997).

Multiple sclerosis (MS) is characterized by a breakdown of the myelinsheath around the nerves and by an influx of leukocytes into the nervoustissue. High levels of chemokines including MIP-1α, RANTES, and MCP-1are found in brain lesions of MS patients; Sorensen, et al., J. Clin.Invest. 103: 807-815 (1999).

Experimental autoimmune encephalitis (EAE) is a disease model thatclosely mimics human MS. MIP-1α and MCP-1 have both been implicated inthe induction of disease symptoms and in development of relapses, asshown with neutralizing antibodies; Kennedy, et al., J. Neuroimmunol.92: 98-108 (1998).

Fibrotic disease includes any condition marked by an increase ofinterstitial fibrous tissue. CC-chemokines are known to be associatedwith fibrotic conditions. For example levels of MCP-1, MIP-1α and MIP-1βare all elevated in patients with systemic sclerosis and high levels ofCC-chemokines correlated with development of lung fibrosis in thesepatients; Hasegawa et al., Clin. Exp. Immunol. 117: 159-165 (1999).

Atherosclerosis is characterized by vascular lipid deposits with highinfiltration of macrophages. MCP-1 knockout mice show a marked decreasein macrophages and lipid deposition in a murine model ofatherosclerosis; Gu, et al., Mol. Cell. 2: 275-281 (1998).

Asthma patients have marked infiltration of leukocytes and increasedlevels of CC-chemokines in the lungs leading to airway hyperresponsiveness. In rodent models of asthma neutralizing antibodies toMIP-1α, MCP-1 or RANTES decreased the inflammation and/or the airwayhyper reactivity typical of this disease; Lukacs, et al., J. Immunol.158: 4398-4404 (1997).

The animal models described in the documents above may be used toevaluate the efficacy of antikine antibodies in treating the associatedcondition, disorder or disease and these models and methods of their useare incorporated by reference to the documents above.

Besides the diseases highlighted above, numerous other conditions,disorders and diseases have been associated with the activity of one ormore chemokines, these include but are not limited to: oncogenicdiseases, inflammatory bowel diseases, atopic dermatitis, psoriasis,stroke, organ transplantation, COPD, glomerulonephritis, lupusnephritis, scleroderma, cirrhosis, Alzheimer's disease, CHF-ischemia,coronary restenosis, diabetic nephropathy/neuropathy/retinopathy,osteoarthritis, periodontitis, yeast and viral infections, anddysregulation of pregnancy. When CC chemokines play a role in thesepathologies, an antikine antibody may be administered to reduce theseverity of these conditions.

Such methods generally involve administering an amount of an antikineantibody usually in a pharmaceutically acceptable carrier to a subject,such as an animal or human, in need thereof. The amount of antikineantibody or a truncated version or fragment thereof is selected so as toinhibit the activity of one or more chemokines in the subject. Similarmethods may be performed ex vivo on biological materials (e.g., blood,bone marrow, organic tissue) removed from a subject or on biologicalmaterials maintained in vitro.

Antikine antibodies may be used to block chemotaxis. Such methodscomprise admixing or contacting an antikine antibody with a mediumcontaining CC chemokines to which the antikine antibody binds or amedium containing cells producing these CC chemokines.

Diagnostic assays can usefully employ the antikine antibodies of theinvention. Such assays involve contacting an antikine antibody havingknown specificities for CC chemokines with a biological sample suspectedof containing at least one chemokine to which the antikine antibodybinds and determining the amount of binding, for example, by measurementof complex formation. The antikine antibodies may be used in free orsubstrate bound form. The invention further provides in vitroimmunoassays for detecting CC-chemokines in samples.

The antibodies and antibody fragments of the invention may be used todetect CC-chemokines in samples using a variety of well-knownimmunological assays. The antibodies may be used, for example, inELISAs, Western blots, radioimmunoassays, immunoprecipitaton,immunoaffinity chromatography, immunostaining of tissue sections,immunogold detection in tissue samples with electron microscopy, and thelike. The protocols for these and other assays are well-known in the artand are well within the purview of the skilled artisan.

The immunoassays using the antibodies and antibody fragments of theinvention may be used to detect the presence and relative amounts ofCC-chemokines in a sample. Samples may include, but are not limited to,homogenized tissue or cells, histological tissue sections for light andelectron microscopy, protein extracts of tissue or cells, csf, jointfluid, blood, plasma, serum, mucosal secretions, semen, vaginal fluids,tears, saliva, sweat, urine, feces and the like. The presence ofincreased amounts of a CC-chemokine(s) relative to normal samples, forexample, may indicate the presence of a disease state, and treatmentwith a therapeutic of the invention may be indicated. In some instances,there may be a decreased amount of CC-chemokine(s) relative to normalsamples, and treatment with appropriate CC-chemokine(s) or internalimage antibodies that mimic CC-chemokine(s) may be used to stimulateimmune function.

In some embodiments, an immunoassay may be used to aid in thepurification of CC-chemokines. For example, an immunoaffinity resin maybe used in which the antibodies or antibody fragments of the inventionare immobilized on a substrate. A sample containing the CC-chemokine(s)is added to the immunoaffinity resin and the antibodies become bound tothe resin, while other components of the sample remain in solution. Theresin is washed and the CC-chemokines are subsequently eluted from theresin, substantially purified and isolated. Preferably, the antibodiesused in the immunoassay will have high binding affinity, as definedherein.

EXAMPLES

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the figures and the Sequence Listing, areincorporated herein by reference.

Example 1 Production of Hybridomas Producing Antikine Antibodies

Mice were immunized sequentially with three CC-chemokines, in randomorder, from the set that initially included MIP-1α, RANTES and MCP-1(PeproTech). For hybridoma fusion 3, which produced MAb 3C12F, the mousewas initially immunized with MIP-1α, followed by boosts with RANTES,then MCP-1, then MCP-1 again. For hybridoma fusion 7, which producedMAbs 7D12A and 7D1G, the mouse was initially immunized with MIP-1α,followed by boosts with MCP-1, then RANTES, then a combination of MIP-1αand RANTES. For hybridoma fusion 18, which produced MAbs 18V4F and18P7E, mice were immunized initially with MIP-1β, followed by boostswith MIP-1α, then RANTES, then another boost with RANTES.

For each immunization 10 μg protein was used, following standardimmunization protocols, see Current Protocols in Immunology, Eds. JEColigan, et al. (2006), section 2.1. The initial immunizations were donein Complete Freund's Adjuvant, followed by 3 week interval boosts of twodifferent chemokines in Incomplete Freund's Adjuvant.

Ten days after the final boost serum was collected and tested forreactivity with CC-chemokines MIP-1α, RANTES, MCP-1 and MIP-1β by ELISAas described by Current Protocols in Immunology, 2006, Eds. JE Coliganet al., section 2.4, which is incorporated by reference. For MIP-1α, thechemokine was coated on a 96-well ELISA plate (at 1 μg/mL) and incubatedwith serum at a range of dilutions from 1:50 to 1:6400. For RANTES,MCP-1 and MIP-1β, biotinylated chemokines (0.5 μg/mL) were added tostreptavidin-coated plates (Pierce) and then incubated with dilutedsera. Antibody bound to coated chemokines was detected using ananti-mouse Fc secondary antibody conjugated to HRP.

Biotinylation of chemokines for ELISA assays was performed usingsulfo-NHS-LC-biotin (Pierce). Chemokine in PBS was mixed withapproximately 2-fold molar excess of sulfo-NHS-LC-biotin and incubatedat room temperature for 30 min. Free biotin reagent was removed bydialysis in PBS overnight using Slide-a-lyzer mini dialysis units with3,500 MW cutoff (Pierce).

Results of ELISAs on serum from the initial set of immunizations usingMIP-1α, RANTES and MCP-1 showed a variety of responses (see Table 1),including some responses to MIP-1β even though it was not used as adirect immunogen. Several mice had responses to 3 and even 4 chemokines.These were candidates for hybridoma fusions to look for singleantibodies that reacted with multiple chemokines. As shown in Table 1below, immunized mice produced polyclonal sera which reacted withmultiple CC chemokines.

TABLE 1 Serum reactivities of immunized mice Immunizatio

Mouse MCP-1 RANTE

MIP-1α MIP-1β sequence A1 + MIP-1α → A2 + RANTES → A3 + + MCP-1 A4 + + +A5 + + + + B1 + + + + MIP-1α → B2 + +/− MCP-1 → B3 + +/− + +/− RANTESB4 + + B5 + + C1 + + + +/− RANTES → C2 +/− + + MIP-1α → C3 + +/− MCP-1C4 + +/− C5 + + D1 + RANTES → D2 + MCP-1 → D3 + + + MIP-1α D4 + + D5 +E1 + + MCP-1 → E2 + + + +/− MIP-1α → E3 + + RANTES E4 + +/− +/− E5 + + +F1 + + + MCP-1 → F2 +/− RANTES→ F3 MIP-1α F4 + +/− F5 +

indicates data missing or illegible when filed

Mice showing significant serum reactivity with at least 3 targetchemokines were selected for hybridoma fusions; see Antibodies: ALaboratory Manual, Eds. E. Harlow & D. Lane (1988), chapter 6.

A chosen mouse was boosted with a mixture of chemokines (20 μg each) inPBS at days −4 and −3 before harvesting the spleen for fusion with NS1cells to create hybridomas. A single cell suspension was made from thespleen by pressing between the frosted ends of two slides and collectingthe cells in 10 mL RPMI. Cells were passed through a 70 μm nylonstrainer, rinsed with 10 mL additional RPMI, and pelleted at 300×g for 5min. Cells were washed and centrifuged 2 more times and then counted.Spleen cells were combined with NS1 cells (ATCC) at a ratio of 5:1spleen:NS1 and then pelleted. One mL 50% PEG was added dropwise to thepelleted cells over 1 min with swirling, then the mixture was swirled anadditional 1 min. An additional 1 mL RPMI was added over 1 min withswirling, then another 3 mL RPMI was added over 1 min with swirling,then another 16 mL RPMI was added over 2 min with swirling. Cells werepelleted at 300×g for 10 min and resuspended in 800 mL hybridomaselection medium. The cells were rested for 2 hours and then distributedover 40 96-well plates at 200 μL/well. Plates were placed in 37° C.incubator and fed every 2-3 days over the next 8-10 days by removing andreplacing half the volume of medium. Alternatively, fused cells weresuspended in methylcellulose-based semi-solid medium CloneMatrix(Genetix) containing CloneDetect (Genetix) plus Alexa488-tagged MIP-1αat 4 μg/mL and plated in single-well 10 cm plates (Genetix).

Example 2 Identification of Antikine Antibodies

Antibodies in the hybridoma supernatant obtained in Example 1 weretested for their ability to recognize MIP-1α, RANTES, MCP-1 and MIP-1βby ELISA (similar to serum tests described above). ELISA results forinitial hybridoma supernatants are shown in Table 2 below. Cells fromfusion wells that showed reactivity of at least 4-fold over backgroundwith more than one chemokine were expanded into 24-well plates forfurther testing. Cells from wells that reacted with at least 3chemokines were cloned by limiting dilution or by serial dilution atleast two times. Clone plate supernatants were again tested by ELISA toidentify individual clones that produced antibody reactive againstmultiple chemokines. Positive wells were confirmed to be clonal byvisual inspection.

Alternatively, hybridomas grown as colonies in semi-solid media wereanalyzed after 16 days for growth and fluorescent chemokine bindingusing ClonePixFL (Genetix), and the best colonies were picked into96-well plates. Antibody-containing supernatants from the hybridomaclones were analyzed after 4 days for reactivity by ELISA with MIP-1α(both directly coated and biotinylated), RANTES and MIP-1β as describedabove for serum analysis. Clones 18V4F and 18P7E showed significantreactivity with all 3 CC-chemokines. These were re-cloned by serialdilution to confirm clonality and again tested by ELISA to confirmproduction of antikine antibodies exhibiting multi-chemokine reactivity.

TABLE 2 ELISA signals from original hybridoma fusion wells for antikinemonoclonal antibodies. a) ELISA signals for 3C12F, 7D1G, and 7D12Aantibodies Backgroun

3C12F 7D1G 7D12A (approx.) MIP-1α 1.84 1.70 1.47 0.150 Biotin-RANTES2.61 0.145 0.178 0.150 Biotin-MCP1 0.189 0.220 0.246 0.150 Biotin-MIP-1β1.92 2.57 2.68 0.150 b) ELISA signals for 18V4F and 18P7E antibodies18V4F 18P7E Background Biotin-MIP-1α 1.674 1.001 0.040 Biotin-RANTES0.909 0.462 0.030 Biotin-MIP-1β 1.699 1.603 0.075

indicates data missing or illegible when filed

Example 3 Characterization of Chemokine Binding Specificities ofAntikine Antibodies

A more complete analysis of the chemokine binding specificities for theisolated antikine antibodies was performed using an MSD (Meso ScaleDiscovery) assay. This is very similar to the ELISA assays done duringantibody screening, however it uses electrochemiluminescence detectionof SULFO-TAG reagents. In this case, a selection of chemokines is coatedat 1 μg/mL on an MSD 96-well multiarray plate (MA2400). Antikineantibodies are added either at 3 μg/mL (purified antibodies 3C12F, 7D12A, 7D 1 G, 18V4F) or as unquantitated hybridoma antibody-containingsupernatant (18P7E) and are detected with a SULFO-TAG anti-murineantibody used at 1 μg/mL. Plates are read on an MSD sector imager 2400.Background levels shown as dotted lines in the Figures are based onbinding to a non-reactive protein, bovine serum albumin (BSA).

Example 4 Examination of the Functional Activities of AntikineAntibodies

Supernatants from expanded fusion wells obtained in Example 2 weretested for ability to block chemotaxis mediated by a target chemokine.This was tested in vitro using CCR2 transfectants (receptor for MCP-1)or CCR5 transfectants (receptor for MIP-1α, RANTES and MIP-1β) in96-well transwell plates (Millipore). In the lower well of eachtranswell chamber, 75 μL of chemokine solution (10 ng/mL in RPMI with 2%FBS) was mixed with 75 μL hybridoma supernatant. In the upper well ofeach chamber 4×10⁵ cells were added in 75 μL RPMI with 2% FBS. Cellmigration was allowed to occur for 2 hours at 37° C., then the number ofcells in the lower chamber was quantitated by cell counting using theFACScalibur. Cells from hybridoma wells that showed inhibition of atleast 2 chemokines were then also cloned by serial dilution. Purifiedantibodies were tested similarly for inhibition of chemotaxis at a rangeof concentrations.

Chemokine receptor transfectants used in chemotaxis assays weregenerated using Ba/F3 cells (obtained from the German Collection ofMicroorganisms and Cell Cultures—DSMZ). The open reading frames of humanCCR2 and CCR5 were amplified by PCR (see Molecular Cloning: A LaboratoryManual, Eds. Sambrook et al.) from cDNA clones purchased from Origene.The PCR primers were designed from published sequences. The 5′ primeroverlapped the initiating Met codon and contained an XhoI cloning site.The 3′ primer overlapped the termination codon and contained an XbaIcloning site. The amplified fragment was digested with XhoI and XbaI andinserted into the equivalent sites in the expression plasmid pNEF38(Running Deer & Allison, Biotechnol Prog 20, 880-889, 2004). Ba/F3 cellswere transfected with the expression plasmids by electroporation (Amaxa)and selected in G418. Cells expressing functional receptor were selectedthrough chemotaxis to the cognate chemokine ligands, and migrating cellswere cloned by limiting dilution to obtain a stable cell clone.

Example 5 Identification and Characterization of Antikine Antibodies

Five unique monoclonal antibodies that recognized multiple CC chemokineswere identified and designated 3C12F, 7D1G, 7D12A, 18V4F and 18P7E. Theisotypes of the antibodies were determined using IsoStrips (Roche). The3C12F heavy chain was determined to be a member of murine subgroup IgG1,while the 3C12F light chain was determined to be a member of murine κgroup. The 7D1G heavy chain was determined to be a member of murinesubgroup IgG1, while the 7D1G light chain was determined to be a memberof murine κ group. The 7D12A heavy chain was determined to be a memberof murine subgroup IgG1, while the 7D12A light chain was determined tobe a member of murine κ group. The 18V4F heavy chain was determined tobe a member of murine subgroup IgG2a, while the 18V4F light chain wasdetermined to be a member of murine λ. The 18P7E heavy chain wasdetermined to be IgG1 and its light chain was also found to be murine λ.

MAb 3C12F recognized MIP-1α, RANTES and MIP-1β as shown using purifiedantibody in FIG. 1 and as originally assayed by ELISA (see Table 2a) andblocked the binding of the viral vCCI molecule to RANTES (FIG. 2). Inchemotaxis assays 3C12F inhibited the function of MIP-1α and RANTES,with IC₅₀ values of 3-5 μg/mL when using 5 ng/mL chemokine to inducechemotaxis (FIG. 3). By BIAcore®, the affinity of 3C12F for MIP-1α is 49nM.

Antibodies 7D1G and 7D12A both recognized MIP-1α and MIP-1β when thehybridoma supernatants were assayed by ELISA (Table 2a). These sameantibodies also blocked the functions of both chemokines in chemotaxisassays as shown by FIG. 4 and FIG. 5.

Antibodies 18V4F and 18P7E both recognized MIP-1α, MIP-1β, and RANTESwhen the hybridoma supernatants were assayed by ELISA (Table 2b). Thesame antibodies also blocked the functions (at least partially) ofchemokines MIP-1α, RANTES, and MIP-1β in chemotaxis assays as shown byFIG. 6 and FIG. 7.

Using the MSD platform to analyze the reactivity of purified antikineantibodies (or antibody supernatants from clonal hybridomas, in the caseof 18P7E) with an array of chemokines, it was found that the antikineantibody 3C12F bound MIP-1α, RANTES, MIP-1β, MPIF-1 and HCC-2 at leastfour-fold over background (FIG. 8). Similarly antikine antibody 7D12Abound MIP-1α, RANTES, MIP-1β and MPIF-1 (FIG. 9), and antikine antibody7D1G bound MIP-1α, RANTES, MIP-1β, HCC-1, MPIF-1 and PARC (FIG. 10).Antikine antibodies 18V4F and 18P7E both bound MIP-1α, RANTES, andMIP-1β (FIG. 11 and FIG. 12).

Table 3 below summarizes the binding specificities of these fiveantikine monoclonal antibodies.

TABLE 3 Specificities and functional activities of MAbs 3C12F, 7D1G,7D12A, 18V4F and 18P7E. 3C12F 7D1G 7D12A 18V4F 18P7E Binding (MSD)MIP-1α/CCL3 Yes Yes Yes Yes Yes MIP-1β/CCL4 Yes Yes Yes Yes YesRANTES/CCL5 Yes Yes Yes Yes Yes MCP-1/CCL2 No No No No No Other CC YesYes Yes No No chemokines (MPIF-1, HC

(HCC-1, MPI

(MPIF-1) 2) 1, PARC) muMIP-1α No Yes No No No muMIP-1β No Yes No No NomuRANTES No No No No No Chemotaxis Inhibition MIP-1α/CCL3 InhibitsInhibits Inhibits Inhibits Inhibits MIP-1β/CCL4 — Inhibits InhibitsInhibits Inhibits RANTES/CCL5 Inhibits — — Inhibits Inhibits MCP-1/CCL2— — — — — Other CC — — — — — chemokines vCCI binding Yes (to ND ND ND NDinhibition to a CC- RANTES) chemokine ND = Not determined

indicates data missing or illegible when filed

Example 6 Analysis of Structures Common to CC Chemokines Bound byAntikine Antibodies

The amino acid sequences of the CC-chemokines bound by monoclonalantibodies 7D1G, 7D12A, 3C12F, 18V4F and 18P7E were aligned. As shown inFIG. 13, several areas of homology or having a high degree of structuralsimilarity were identified to which the antikine monoclonal antibodiesof the invention bind. The identification of these common or conservedsegments of CC chemokines characterizes antibody binding and serves ascore structures for production or screening of new antikine antibodies.

FIGS. 13 a-d show the alignments of the CC chemokines bound by eachantikine antibody 3C12F (FIG. 13 a), 7D12A (FIG. 13 b), 7D1G (FIG. 13c), 18V4F (FIGS. 13 d) and 18P7E (FIG. 13 d). Identical amino acidresidues shared by the CC chemokines bound by a particular antikineantibody are shaded. Similar amino acid residues were designatedaccording to Table 5, for example, the amino acid residue alanine (A) issimilar to glycine, serine or threonine.

The shared identical or similar residues were further characterized asnot solvent exposed, partially solvent exposed or fully solvent exposedbased on FIGS. 1 and 2 of Fernandez, id. and Czaplewski et al., J. Biol.Chem. 274:16077-84 (1999), both of which are incorporated by reference.

Further analysis correlated the shared, solvent exposed residues of thealigned identical or identical+similar residues with CC chemokineresidues associated with binding of the CC chemokine receptor. Thestructural information regarding chemokine receptor binding sites andother structural, chemical, or functional information is incorporated byreference to the entries for the chemokines or other biologicalmolecules described herein to the NCBI Conserved Domain Database CDD29111 [uid] and to the web addresses shown below each last accessed Aug.9, 2010.

Putative receptor binding site residues and other structural featuresfor MIP-1α/CCL3 (SEQ ID NO: 71) are described athttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=NP_(—)002974.1.

Putative receptor binding site residues and other structural featuresfor MIP-1β/CCL4 (SEQ ID NO: 72) are described athttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=AAH70310.1.

Putative receptor binding site residues and other structural featuresfor RANTES/CCL5 (SEQ ID NO:73) are described athttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=P13501.3.

Putative receptor binding site residues and other structural featuresfor MPIF-1/CCL23 (SEQ ID NO:81) are described athttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=P55773.2.

Putative receptor binding site residues and other structural featuresfor HCC-1/CCL14 (SEQ ID NO: 78) are described athttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=NP_(—)004157.1.

Putative receptor binding site residues and other structural featuresfor HCC-2/CCL15 (SEQ ID NO: 79) are described athttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=Q16663.2.

Putative receptor binding site residues and other structural featuresfor PARC/CCL18 (SEQ ID NO:82) are described at http://www.nebi.nlmnih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=P55774.1.

MAb 3C12F recognizes CC chemokines that share identical amino acidresidues C, C, Y, P, Y, T, C, S, P, V, F, T, C, A, P, V and L atrelative positions 11, 12, 15, 21, 28, 31, 35, 36, 38, 40, 42, 44, 51,52, 54, 59 and 66 in FIG. 13 a. Of these residues, residues 11, 12, 15,21, 28, 31, 35, 36, 38, 54 and 66 are partially or fully solvent exposedbased on FIGS. 1 and 2 of Fernandez, id and Czaplewski, id.

In addition, the CC chemokines recognized by MAb 3C12F share chemicallysimilar amino acid residues at positions 9, 14, 20, 25, 29, 33, 39, 41,45, 46, 48 and 63 in FIG. 13 a. Of these residues, those at 14, 20, 29,33, 39, 45, 46, and 48 are partially of fully exposed to solvent(compare FIG. 13 a and FIGS. 1 and 2 of Fernandez, et al.).

Based on this structural analysis, identical and similar residues 11,12, 14, 15, 20, 21, 28, 29, 31, 33, 35, 36, 38, 39, 45, 46, 48, 54 and66 form shared portions of the CC chemokine molecules bound by MAb 3C12Favailable as determinants for binding between this antibody and the CCchemokines it recognizes.

Of these shared, solvent-exposed residues, residues 14, 15, 20, 21 and66 have been characterized as chemokine receptor contact residuesaccording to NCBI Conserved Domain Database CDD 29111. Blocking orbinding of an antikine antibody to contact residues for a CC chemokinereceptor would affect or modulate the ability of a CC chemokine to bindto its receptors.

The shared, solvent-accessible residues of CC chemokines bound by MAb3C12F have been further correlated with CC chemokine structure andappear in the N-loop between the initial CC residues of the CCchemokines and the β1 strand, as well as in the 30's loop of the CCchemokines between the β1 and β2 strands.

MAb 7D12A recognizes CC chemokines with amino acid residues C, C, Y, R,P, Y, T, C, S, P, V, F, T, C, A, P, V and L at relative positions 11,12, 15, 18, 21, 28, 31, 35, 36, 38, 40, 42, 44, 51, 52, 54, 59 and 66 inFIG. 13 b. Of these residues, residues 11, 12, 15, 18, 21, 28, 31, 35,36, 38, 54 and 66 are partially or fully solvent exposed based on FIGS.1 and 2 of Fernandez, id and Czaplewski, id.

In addition, the CC chemokines recognized by MAb 7D12A share similaramino acid residues at positions 9, 14, 20, 25, 29, 33, 39, 41, 45, 46,48 and 63 in FIG. 13 b. Of these residues, those at 14, 20, 29, 33, 39,45, 46 and 48 are partially or fully exposed to solvent (compare FIG. 13b and FIGS. 1 and 2 of Fernandez, et al.).

Based on this structural analysis, identical and similar residues 14,15, 18, 20, 21, 28, 29, 31, 33, 35, 36, 38, 39, 45, 46, 48, 54 and 66form shared portions of the CC chemokine molecules bound by MAb 7D12Aavailable as determinants for binding between this antibody and the CCchemokines it recognizes.

Of these residues, residues 14, 15, 18, 20, 21 and 66 have beencharacterized as chemokine receptor contact residues according to NCBIConserved Domain Database CDD 29111. Competitive antibody bindingencompassing these residues would affect the ability of a CC chemokineto bind to its receptors. For CC chemokines recognized by MAb 7D12Aseveral shared solvent accessible residues appear in the N-loop betweenthe initial CC residues of the CC chemokines and the 131 strand, in the30's loop of the CC chemokines between the β1 and β2 strands, as well asin the 40's loop between the β2 and β3 strands.

MAb 7D1G recognizes CC chemokines with identical amino acid residues C,C, Y, P, Y, T, C, P, T, C, P, and V at relative positions 11, 12, 15,21, 28, 31, 35, 38, 44, 51, 54 and 59 in FIG. 13 c. Of these residues,residues 11, 12, 15, 21, 28, 31, 35, 38 and 54 are partially or fullysolvent exposed based on FIGS. 1 and 2 of Fernandez, id and Czaplewski,id.

In addition, the CC chemokines recognized by MAb 7D1G share similaramino acid residues at positions 20, 25, 39-41, 45, 46, 63 and 66 inFIG. 13 c. Of these residues, those at 20, 39, 45, 46 and 66 arepartially or fully exposed to solvent (compare FIG. 13 c and FIGS. 1 and2 of Fernandez, et al.).

Based on this structural analysis, identical and similar residues 11,12, 15, 20, 21, 28, 31, 35, 38, 39, 45, 46, 54, and 66 form sharedportions of the CC chemokine molecules bound by MAb 7D1G available asdeterminants for binding between this antibody and the CC chemokines itrecognizes.

Of these shared residues, residues 15, 20, 21, 24 and 66 have beencharacterized as chemokine receptor contact residues according to NCBIConserved Domain Database CDD 29111. Competitive antibody bindingencompassing these residues would affect the ability of a CC chemokineto bind to its receptors. For CC chemokines recognized by MAb 7D1Gseveral shared solvent accessible residues appear in the N-loop betweenthe initial CC residues of the CC chemokines and the β1 strand, as wellas in the 40's loop between the β2 and β3 strands.

MAb 18V4F and MAb 18P7E recognize CC chemokines with amino acid residuesC, C, Y, R, P, Y, T, C, S, P, V, F, T, C, A, P, V and L at relativepositions 11, 12, 15, 18, 21, 28, 31, 35, 36, 38, 40, 42, 44, 51, 52,54, 59 and 66 in FIG. 13 d. Of these residues, residues 11, 12, 15, 18,21, 28, 31, 35, 36, 38, 54 and 66 are partially or fully solvent exposedbased on FIGS. 1 and 2 of Fernandez, id and Czaplewski, id.

In addition, the CC chemokines recognized by MAb 18V4F and 18P7E sharesimilar amino acid residues at positions 9, 14, 20, 25, 29, 33, 39, 41,45, 46, 48 and 63 in FIG. 13 d. Of these residues, those at 14, 20, 29,33, 39, 45, 46 and 48 are partially or fully exposed to solvent (compareFIG. 13 d and FIGS. 1 and 2 of Fernandez, et al.).

Based on this structural analysis, identical and similar residues 14,15, 18, 20, 21, 28, 29, 31, 33, 35, 36, 38, 39, 45, 46, 48, 54 and 66form shared portions of the CC chemokine molecules bound by MAb 18V4Fand 18P7E available as determinants for binding between these antibodiesand the CC chemokines they recognize.

Of these residues, residues 14, 15, 18, 20, 21 and 66 have beencharacterized as chemokine receptor contact residues according to NCBIConserved Domain Database CDD 29111. Competitive antibody bindingencompassing these residues would affect the ability of a CC chemokineto bind to its receptors. For CC chemokines recognized by MAb 18V4F and18P7E several shared solvent accessible residues appear in the N-loopbetween the initial CC residues of the CC chemokines and the β1 strand,in the 30's loop of the CC chemokines between the β1 and β2 strands, aswell as in the 40's loop between the β2 and β3 strands.

Example 7 Polynucleotide Sequences of Antikine Antibodies

The sequences of the variable domains of the antibodies were determinedafter PCR amplification of these regions using primers within theconstant regions. RNA was extracted from 10⁷ hybridoma cells usingRNeasy (Qiagen), then reverse transcribed using the SuperScript IIIReverse Transcription kit from Invitrogen. The CDR regions wereamplified by PCR using a set of primers for either the light chain orthe heavy chain (Jones & Bendig, Biotechnology 9, 88-89, 1991). Theresulting fragments were inserted into the pCR11 plasmid using the ZeroBlunt TOPO PCR cloning kit from Invitrogen and sequenced using M13 andM13rev primers. Polynucleotide sequences encoding light and heavy chainsegments, such as CDRs, of monoclonal antibodies 3C12F, 7D1G, 7D12A,18V4F and 18P7E are described below. The DNA coding sequence of the3C12F heavy chain variable region is set forth as SEQ ID NO: 1 and theamino acid sequence of the 3C12F heavy chain variable region is setforth as SEQ ID NO: 2. The DNA coding sequence of the 3C12F light chainvariable region is set forth as SEQ ID NO: 6 and the amino acid sequenceof the light chain variable region of 3C12F is set forth as SEQ ID NO:7. Furthermore, the CDRs of antibody 3C12F are described as SEQ ID NOs:3-5 (heavy chain variable region) and SEQ ID NOs: 8-10 (light chainvariable region).

The DNA coding sequence of the 7D12A heavy chain variable region is setforth as SEQ ID NO: 11 and the amino acid sequence of the 7D12A heavychain variable region is set forth as SEQ ID NO: 12. The DNA codingsequence of the 7D12A light chain variable region is set forth as SEQ IDNO: 16 and the amino acid sequence of light chain variable region of7D12A is set forth as SEQ ID NO: 17. Furthermore, the CDRs of antibody7D12A are described as SEQ ID NOs: 13-15 (heavy chain variable region)and SEQ ID NOs: 18-20 (light chain variable region).

The DNA coding sequence of the 7D1G heavy chain variable region is setforth as SEQ ID NO: 21 and the amino acid sequence of the 7D1G heavychain variable region is set forth as SEQ ID NO: 22. The DNA codingsequence of the 7D1G light chain variable region is set forth as SEQ IDNO: 26 and the amino acid sequence of light chain variable region of7D1G is set forth as SEQ ID NO: 27. Furthermore, the CDRs of antibody7D1G are described as SEQ ID NOs: 23-25 (heavy chain variable region)and SEQ ID NOs: 28-30 (light chain variable region).

The DNA coding sequence of the 18V4F heavy chain variable region is setforth as SEQ ID NO: 31 and the amino acid sequence of the 18V4F heavychain variable region is set forth as SEQ ID NO: 32. The DNA codingsequence of the 18V4F light chain variable region is set forth as SEQ IDNO: 36 and the amino acid sequence of the light chain variable region of18V4F is set forth as SEQ ID NO: 37. Furthermore, the CDRs, as definedby Chothia and Lesk, of antibody 18V4F are described as SEQ ID NOs:33-35 (heavy chain variable region) and SEQ ID NOs: 38-40 (light chainvariable region).

The DNA coding sequence of the 18P7E heavy chain variable region is setforth as SEQ ID NO: 41 and the amino acid sequence of the 18P7E heavychain variable region is set forth as SEQ ID NO: 42. The DNA codingsequence of the 18P7E light chain variable region is set forth as SEQ IDNO: 46 and the amino acid sequence of the light chain variable region of18P7E is set forth as SEQ ID NO: 47. Furthermore, the CDRs, as definedby Chothia and Lesk, of antibody 18P7E are described as SEQ ID NOs:43-45 (heavy chain variable region) and SEQ ID NOs: 48-50 (light chainvariable region).

Example 8 Humanization of Monoclonal Antibody 3C12F

Antibody humanization methods are designed to produce a molecule withreduced human immunogenicity. Methods of antibody humanization are wellknown to those of ordinary skill in the art and include those describedby Almagro and Frannson, Frontiers Bioscience 13:1619-1633 (2008). TheCDR grafting method (Jones et al., Nature 321:522-525, 1986; Riechmann,et al., Nature 332:323-327, 1988) was used to embed the CDR sequences of3C12F into heavy and light chain IgG variable region framework sequencesfrom similar human germline genes. In this case, the rodent CDRsequences have been embedded into heavy and light chain IgG variableregion framework sequences from chosen human germline genes that havethe highest identity to the respective rodent heavy and light chainvariable sequences. See Jones et al., Nature 321:522-525 (1986);Riechmann, et al., Nature 332:323-327 (1988), which are incorporatedherein in their entirety, for suitable methods.

In humanizing 3C12F, the human germline sequences for the variable heavyand light chains that shared the greatest similarity with murine 3C12Fsequence were used as the human framework sequences. In addition tosharing high sequence similarity with the murine antibody, the chosengermline sequences were also found to be abundant in a sampling ofrearranged antibody sequences. The murine residues that reside in thecomplementary determining regions (CDRs) as defined by Kabat weregrafted into the human framework sequences with additional residuesbeing reverted to the murine amino acid when evidence indicated thatthey may be essential for binding. The polynucleotide sequences encodingthe heavy and light chains of the humanized 3C12F MAb are given by SEQID NOS: 51 and 56. The corresponding amino acid sequences and CDRs areidentified by SEQ ID NOS: 52-55 and 57-60.

Example 9 Humanization of Monoclonal Antibody 18V4F

In humanizing 18V4F, a different strategy was employed than that used inhumanizing 3C12F. Instead of grafting the CDRs of the murine 18V4Fantibody into the most similar human germline sequence, the CDRs weregrafted into a consensus sequence compiled from the most populatedvariable heavy chain family, VHIII, and the most populated variablelambda light chain family, VLλIII. Next, only those residues in the CDRthat have been defined by Chothia and Lesk to be structurally relevantfor binding were initially grafted into the consensus frameworks.Additional CDR positions defined more broadly by Kabat, as well asframework residues, were reverted back the murine amino acid whenevidence indicated they may be important to retain binding. Thepolynucleotide sequences encoding the heavy and light chains of thehumanized 18V4F MAb are given by SEQ ID NOS: 61 and 66. Thecorresponding amino acid sequences and CDRs are identified by SEQ IDNOS: 62-65 and 67-70.

Example 10 Affinity Maturation of Humanized MAb 3C12F and Mab 18V4F

The binding affinities of the antikine monoclonal antibodies of theinvention may be further enhanced, if desired, by affinity maturation asdescribed for example by Wu et al. (1998), Proc. Natl. Acad. Sci. USA95: 6037-6042, which is hereby incorporated by reference.

To improve the binding characteristics of humanized MAb 3C12F andhumanized MAb 18V4F to various chemokines (e.g., RANTES/CCL5,MIP-1α/CCL3 and MIP-1β/CCL4) and to generate more potent agents, asite-directed mutagenesis strategy in conjunction with phage display wasemployed. The Fab fragment of humanized 3C12F or humanized 18V4F wasfirst displayed on the surface of filamentous phage M13 as a heavy chainfusion with the M13 gene III protein with co-expression of the humanized3C12F or humanized 18V4F light chain, respectively. Next, every positionof all six CDRs of humanized Mab 3C12F or humanized 18V4F wasmethodically mutated, 4-6 contiguous CDR residues at a time, using theprotocol of Sidhu and Weiss (Phage Display, A Practical Approach,Clackson, T. and Lowman, H. B., ed., Oxford University Press, 2004,Chapter 4). In some cases, the amino acids surrounding the defined CDRswere also mutated in case they were involved in antigen binding. A totalof 15 libraries were constructed for each antibody.

Phage displaying mutant humanized 3C12F or humanized 18V4F Fab fragmentswere prepared for each library for binding selections. Up to six roundsof phage selection for higher affinity variants of humanized 3C12F orhumanized 18V4F were conducted using the affinity maturation protocolsof Nielsen and Marks (Clackson and Lowman, ibid, Chapter 14). Briefly,binding selections are undertaken using biotinylated chemokines andcaptured using streptavidin-coated magnetic beads to identify highervariants of humanized 3C12F Fab or humanized 18V4F Fab. Higher affinityvariants are first identified using methods well known in the art (seeClackson and Lowman and references cited therein) and then combined toachieve greater affinity improvements.

The affinity of antibodies for their targets also can be increased usingother various methods known in the art. Affinity can be increased bydirect mutation, phage display, or chain shuffling within the nucleicacids encoding the antibody molecules. Individual or multiple residuescan be randomized so that in a population of otherwise identical antigenantibody sites, all twenty amino acids or a subset of these are found atparticular positions within or adjacent to the CDRs. Useful methods forthis purpose are incorporated by reference to Yang et al., J. Mol. Biol.254, 392-403 (1995); Hawkins et al., J. Mol. Biol. 226, 889-896 (1992);or Low et al., J. Mol. Biol. 250, 359-368 (1996). Marks et al.,Bio/Technology, 10:779-783 (1992) describes affinity maturation by VHand VL domain shuffling; random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad. Sci., USA91:3809-3813 (1994); Schier et al., Gene, 169:147-155, 1995, Yelton etal., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol.154(7):3310-3319 (1995); and Hawkins et al., J. Mol. Biol., 226:889-896(1992) each of which is also incorporated by reference.

Affinity maturation is sometimes carried out using random mutagenesis.Amino acid substitutions made at particular positions can be random, ormade using simplistic rules. For example, all residues could be mutatedto alanine, which is referred to as alanine scanning, see e.g., WO9523813. Sequence-based methods of affinity maturation may also be usedto increase the binding affinities of antibodies, see U.S. 2003/022240A1 and U.S. 2002/177170A1 both of which are incorporated by reference.Other methods include oligonucleotide-based mutagenesis of amino acidswithin some or all positions with some or all CDRs, followed byscreening for higher affinity, as described by Wu et al., Proc. Natl.Acad. Sci. U.S.A., 95, 6037-6042 (1998) or by selection for higheraffinity using phage display, as described by Rajpal, et al., Proc.Natl. Acad. Sci. U.S.A., 102:8466-8471 (1996). Selection of affinitymatured antibodies can also be done by displaying the antibody or afragment thereof on the surface yeast cells, as described by Siegal,Methods Mol. Biol., 504:351-83 (2009), or on the surface of mammaliancells, as described by Ho, et al., Proc. Natl. Acad. Sci. U.S.A.,103:9637-9642 (2006). The methods described in the documents above whichmay be used in the process of affinity maturation are each incorporatedby reference to the documents cited in this Example.

Identification of CC Chemokine and Cowpox Virus Sequences

The identities of the chemokines and other products disclosed herein areincorporated by reference to the accession numbers in Table 4.

TABLE 4  Accession Numbers for CC Chemokines and Other Molecules SEQCatalog NCBI Chemokine ID # Accession # Amino Acid Sequence HUMANMIP-1α/ 71 Pepro P10147.1; ASLAADTPTACCFSYTSRQIPQNFI CCL3 TechNP_002974.1 ADYFETSSQCSKPGVIFLTKRSRQV 300-08 CADPSEEWVQKYVSDLELSAMIP-1β/ 72 Pepro GenBank APMGSDPPTACCFSYTARKLPHNFVV CCL4 Tech AAH70310.1DYYETSSLCSQPAVVFQTKRGKQVCA 300-09 DPSESWVQEYVYDLELN RANTES/ 73 PeproP13501.3 SPYSSDTTPCCFAYIARPLPRAHIKEYF CCL5 TechYTSGKCSNPAVVFVTRKNRQVCANPE 300-06 KKWVREYINSLEMS MCP-1/ 74 PeproP13500.1 QPDAINAPVTCCYNFTNRKISVQRLAS CCL2 TechYRRITSSKCPKEAVIFKTIVAKEICADP 300-04 KQKWVQDSMDHLDKQTQTPKT MCP-2/ 75R & D P80075.2 QPDSVSIPITCCFNVINRKIPIQRLESYT CCL8 SystemsRITNIQCPKEAVIFKTKRGKEVCADPK 281-CP ERWVRDSMKHLDQIFQNLKP MCP-3/ 76 R & DP80098.3 QPVGINTSTTCCYRFINKKIPKQRLESY CCL7 SystemsRRTTSSHCPREAVIFKTKLDKEICADP 282-P3 TQKWVQDFMKHLDKKTQTPKL MCP-4/ 77 R & DQ99616.1 QPDALNVPSTCCFTFSSKKISLQRLKS CCL13 SystemsYVITTSRCPQKAVIFRTKLGKEICADP 327-P4 KEKWVQNYMKHLGRKAHTLKT HCC-1/ 78 R & DNP_004157.1 GPYHPSECCFTYTTYKIPRQRIMDYYE CCL14/ SystemsTNSQCSKPGIVFITKRGHSVCTNPSDK CCL14a 1578-HC WVQDYIKDMKEN HCC-2/ 79 R & DQ16663.2 SFHFAADCCTSYISQSIPCSLMKSYFET MIP-1δ/ SystemsSSECSKPGVIFLTKKGRQVCAKPSGPG CCL15 628-LK VQDCMKKLKPYSI HCC-4/ 80 R & DO15467.1 QPKVPEWVNTPSTCCLKYYEKVLPR CCL16 SystemsRLVVGYRKALNCHLPAIIFVTKRNR 802-HC EVCTNPNDDWVQEYIKDPNLPLLPTRNLSTVKIITAKNGQPQLLNSQ MPIF-1/ 81 R & D P55773.2RFHATSADCCISYTPRSIPCSLLESYF CCL23 Systems ETNSECSKPGVIFLTKKGRRFCANPS131-M1 DKQVQVCMRMLKLDTRIKTRKN PARC/ 82 R & D P55774.1AQVGTNKELCCLVYTSWQIPQKFIV CCL18 Systems DYSETSPQCPKPGVILLTKRGRQICA394-PA DPNKKWVQKYISDLKLNA Eotaxin/ 83 R & D P51671.1;GPASVPTTCCFNLANRKIPLQRLESY CCL11 Systems Q6I9T4RRITSGKCPQKAVIFKTKLAKDICAD 320-EO PKKKWVQDSMKYLDQKSPTPKP Eotaxin-2/ 84R & D GenBank VVIPSPCCMFFVSKRIPENRVVSYQL CCL24 Systems AAB51135.1SSRSTCLKGGVIFTTKKGQQFCGDP 343-E2 KQEWVQRYMKNLDAKQKKASPRARAVAVKGPVQRYPGNQTTC Eotaxin3/ 85 R & D Q9Y258.1 TRGSDISKTCCFQYSHKPLPWTWVCCL26 Systems RSYEFTSNSCSQRAVIFTTKRGKKV 346-E3 CTHPRKKWVQKYISLLKTPKQLMDC/ 86 R & D O00626.1 GPYGANMEDSVCCRDYVRYRLPL CCL22 SystemsRVVKHFYWTSDSCPRPGVVLLTFR 336-MD DKEICADPRVPWVKMILNKLSQ TARC/ 87 R & DQ92583.1 ARGTNVGRECCLEYFKGAIPLRKL CCL17 Systems KTWYQTSEDCSRDAIVFVTVQGR364-DN AICSDPNNKRVKNAVKYLQSLERS LARC/ 88 R & D P78556.1ASNFDCCLGYTDRILHPKFIVGFTR MIP-3α/ Systems QLANEGCDINAIIFHTKKKLSVCANCCL20 360-MP PKQTWVKYIVRLLSKKVKNM ELC/ 89 R & D Q99731.1GTNDAEDCCLSVTQKPIPGYIVRNF MIP-3β/ Systems HYLLIKDGCRVPAVVFTTLRGRQL CCL19361-MI CAPPDQPWVERIIQRLQRTSAKMK RRSS SLC/6Ckine/ 90 R & D O00585.1;SDGGAQDCCLKYSQRKIPAKVVRS CCL21 Systems Q6ICR7 YRKQEPSLGCSIPAILFLPRKRSQAE366-6C LCADPKELWVQQLMQHLDKTPSPQ KPAQGCRKDRGASKTGKKGKGSK GCKRTERSQTPKGPI-309/ 91 R & D P22362.1 KSMQVPFSRCCFSFAEQEIPLRAILC CCL1 SystemsYRNTSSICSNEGLIFKLKRGKEACAL 272-I DTVGWVQRHRKMLRHCPSKRK TECK/ 92 R & DGenBank QGVFEDCCLAYHYPIGWAVLRRAW CCL25 Systems AAB69981.1TYRIQEVSGSCNLPAAIFYLPKRHRK 334-TK VCGNPKSREVQRAMKLLDARNKVFAKLHHNMQTFQAGPHAVKKLSSGN SKLSSSKFSNPISSSKRNVSLLISANS GL CTACK/ 93 R & DQ9Y4X3.1 FLLPPSTACCTQLYRKPLSDKLLRKV CCL27 SystemsIQVELQEADGDCHLQAFVLHLAQRS 376-CT ICIHPQNPSLSQWFEHQERKLHGTLP KLNFGMLRKMGCCL28 94 R & D Q9NRJ3.1 ILPIASSCCTEVSHHISRRLLERVNMC SystemsRIQRADGDCDLAAVILHVKRRRICVS 717-VC PHNHTVKQWMKVQAAKKNGKGNVCHRKKHHGKRNSNRAHQGKHETYG HKTPY XCL1 95 R & D P47992.1MVGSEVSDKRTCVSLTTQRLPVSRI Systems KTYTITEGSLRAVIFITKRGLKVCAD 695-LTPQATWVRDVVRSMDRKSNTRNNM IQTKPTGTQQSTNTAVTLTG Fractalkine/ 96 R & DGenBank MAPISLSWLLRLATFCHLTVLLAGQ CX3CL1 Systems AAB49679.1HHGVTKCNITCSKMTSKIPVALLIH 365-FR YQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTR NGGTFEKQIGEVKPRTTPAAGGMDESVVLEPEATGESSSLEPTPSSQEAQ RALGTSPELPTGVTGSSGTRLPPTPKAQDGGPVGTELFRVPPVSTAATWQ SSAPHQPGPSLWAEAKTSEAPSTQDPSTQASTASSPAPEENAPSEGQRVW GQGQSPRPENSLEREEMGPVPAHTDAFQDWGPGSMAHVSVVPVSSEGTP SREPVASGSWTPKAEEPIHATMDPQ RLGVLITPVPDAQAATRIL-8/ 97 R & D P10145.1 SAKELRCQCIKTYSKPFHPKFIKELR CXCL8 SystemsVIESGPHCANTEIIVKLSDGRELCLD 208-IL PKENWVQRVVEKFLKRAENS Gro-α/ 98 R & DP09341.1 ASVATELRCQCLQTLQGIHPKNIQS CXCL1 SystemsVNVKSPGPHCAQTEVIATLKNGRK 275-GR ACLNPASPIVKKIIEKMLNSDKSN Gro-β/ 99 R & DP19875.1 APLATELRCQCLQTLQGIHLKNIQS CXCL2 SystemsVKVKSPGPHCAQTEVIATLKNGQK 276-GB ACLNPASPMVKKIIEKMLKNGKSN Gro-γ/ 100R & D P19876.1 ASVVTELRCQCLQTLQGIHLKNIQS CXCL3 SystemsVNVRSPGPHCAQTEVIATLKNGKK 277-GG ACLNPASPMVQKIIEKILNKGSTN ENA-78/ 101R & D P42830.1 AGPAAAVLRELRCVCLQTTQGVHP CXCL5 SystemsKMISNLQVFAIGPQCSKVEVVASLK 254-X NGKEICLDPEAPFLKKVIQKILDGG NKEN GCP-2/102 R & D P80162.4 VSAVLTELRCTCLRVTLRVNPKTIG CXCL6 SystemsKLQVFPAGPQCSKVEVVASLKNGK 333-GC QVCLDPEAPFLKKVIQKILDSGNK KN NAP-2/ 103R & D P02775.3 AELRCMCIKTTSGIHPKNIQSLEVIG CXCL7 SystemsKGTHCNQVEVIATLKDGRKICLDP 393-NP DAPRIKKIVQKKLAGDESAD PF4/ 104 R & DP02776.2 EAEEDGDLQCLCVKTTSQVRPRHI CXCL4 SystemsTSLEVIKAGPHCPTAQLIATLKNGR 795-P4 KICLDLQAPLYKKIIKKLLES IP-10/ 105 R & DP02778.2 MVPLSRTVRCTCISISNQPVNPRSL CXCL10 SystemsEKLEIIPASQFCPRVEIIATMKKKG 266-IP EKRCLNPESKAIKNLLKAVSKERSK RSP MIG/ 106R & D Q07325.1 TPVVRKGRCSCISTNQGTIHLQSLK CXCL9 SystemsDLKQFAPSPSCEKIEIIATLKNGVQT 392-MG CLNPDSADVKELIKKTEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSR QKKTT I-TAC/ 107 R & D O14625.1FPMFKRGRCLCIGPGVKAVKVADIE CXCL11 Systems KASIMYPSNNCDKIEVIITLKENKG672-IT QRCLNPKSKQARLIIKKVERKNF SDF-1/ 108 R & D P48061.1;KPVSLSYRCPCRFFESHVARANVK CXCL12 Systems Q9H554 HLKILNTPNCALQIVARLKNNNRQ350-NS VCIDPKLKWIQEYLEKALNK BCA-1/ 109 R & D O43927.1;VLEVYYTSLRCRCVQESSVFIPRRF BLC/ Systems Q53X90 IDRIQILPRGNGCPRKEIIVWKKNKCXCL13 801-CX SIVCVDPQAEWIQRMMEVLRKRS SSTLPVPVFKRKIP CXCL16 110 R & DQ9H2A7.4 NEGSVTGSCYCGKRISSDSPPSVQ Systems FMNRLRKHLRAYHRCLYYTRFQL 976-CXLSWSVCGGNKDPWVQELMSCLD LKECGHAYSGIVAHQKHLLP BRAK/ 111 R & D O95715.1SKCKCSRKGPKIRYSDVKKLEMK CXCL14 Systems PKYPHCEEKMVIITTKSVSRYRGQ 866-CXEHCLHPKLQSTKRFIKWYNAWN EKRRVYEE MOUSE MIP-1α/ 112 R & D P10855.2APYGADTPTACCFSYSRKIPRQFI CCL3 Systems VDYFETSSLCSQPGVIFLTKRNRQ 450-MAICADSKETWVQEYITDLELNA MIP-β/ 113 R & D P14097.3 APMGSDPPTSCCFSYTSRQLHRSFCCL4 Systems VMDYYETSSLCSKPAVVFLTKRG 451-MA RQICANPSEPWVTEYMSDLELNRANTES/ 114 R & D P30882.2 SPYGSDTTPCCFAYLSLALPRAHV CCL5 SystemsKEYFYTSSKCSNLAVVFVTRRNR 478-MR QVCANPEKKWVQEYINYLEMS MCP-1/ 115 R & D81870303; QPDAVNAPLTCCYSFTSKMIPM CCL2 Systems Q5SVU3SRLESYKRITSSRCPKEAVVFVT 479-JE KLKREVCADPKKEWVQTYIKNLDRNQMRSEPTTLFKTASALRS SAPLNVKLTRKSEANASTTFST TTSSTSVGVTSVTVN MCP-5/ 116R & D Q62401.1 GPDAVSTPVTCCYNVVKQKIH CCL12 SystemsVRKLKSYRRITSSQCPREAVIFR 428-P5 TILDKEICADPKEKWVKNSINH LDKTSQTFILEPSCLGCOXPOX VIRUS vCCI 117 N/A NP_620006.1 MKQIVLACICLAAVAIPTSLQQSFSSSSSCTEEENKHHMGIDVII KVTKQDQTPTNDKIC QSVTEV TESEDESEEVVKGDPTTYYTVVGGGLTMDFGFTKCPKISSISE YSDGNTVNARLSSVSPGQGKD SPAITREEALSMIKDCEMSINIKCSEEEKDSNIKTHPVLGSNISH KKVSYEDIIGSTIVDTKCVKNL EISVRIGDMCKESSELEVKDGFKYVDGSASEDAADDTSLINSA KLIACV

TABLE 5 Amino acids of strong similarity Amino Acid Similar Amino Acid AG, S, T D E E D F W, Y, H G A, S, T H Y, F, W I L, M, W K R L I, M, V MI, L, V N Q Q N R K S A, T, G T S, G, A V I, L, M W F, Y, H Y F, H, W

Hybridoma Deposits

Hybridoma cell lines 3C12F, 7D12A, 7D1G, 18V4F, and 18P7E were depositedon Aug. 12, 2010 at the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, USA under the followingaccession numbers:

Mouse Hybridoma ATCC Accession Number 3C12F PTA-11261 7D12A PTA-112597D1G PTA-11257 18V4F PTA-11260 18P7E PTA-11258

Modifications and Other Embodiments

Modifications and variations of the described antikine antibodies andcompositions and methods of their production and use, as well as theconcept of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in relation with specific embodiments,it should be understood that the invention as claimed is not intended tobe limited to such specific embodiments.

INCORPORATION BY REFERENCE

Each document, patent, patent application or patent publication cited byor referred to in this disclosure is incorporated by reference in itsentirety, especially with respect to the specific subject mattersurrounding the citation of the reference in the text. However, noadmission is made that any such reference constitutes background art andthe right to challenge the accuracy and pertinence of the citeddocuments is reserved. In case of a conflict between definitions of aterm, the definition of the term as given in the specification willcontrol.

1.-46. (canceled)
 47. An isolated antibody or antigen binding fragmentthereof that binds to at least three different CC chemokines.
 48. Theisolated antibody or antigen binding fragment thereof of claim 47 thatbinds to CCL3/MIP-1α.
 49. The isolated antibody or antigen bindingfragment thereof of claim 47 that binds to CCL4/MIP-1β.
 50. The isolatedantibody or antigen binding fragment thereof of claim 47 that binds toCCL5/RANTES.
 51. The isolated or purified monoclonal antibody of claim47 that is a chimeric monoclonal antibody or an antigen binding fragmentthereof.
 52. The isolated or purified monoclonal antibody of claim 47that is a humanized monoclonal antibody or an antigen binding fragmentthereof.
 53. The isolated or purified monoclonal antibody of claim 47that is a full-length monoclonal antibody.
 54. An antigen bindingfragment of the isolated or purified monoclonal antibody of claim 47.55. The fragment of claim 54 that is an Fab′ or F(ab′)2.
 56. Themonoclonal antibody or fragment thereof of claim 47 that has beenconjugated to an effector moiety, targeting moiety, heterologousprotein, toxin, enzyme, cytokine, chemical tag, radiological tag, asubstance that increases its biological half-life or a substance thatincreases its biological availability.
 57. A composition comprising (i)the isolated or purified monoclonal antibody or the fragment thereof ofclaim 47 and (ii) a carrier or excipient.
 58. A kit for use in atherapeutic or diagnostic procedure that comprises one more antikineantibodies, at least one positive control antibody, at least onenegative control antibody, at least one CC chemokine bound by said oneor more antikine antibodies, at least one control chemokine not bound bythe antikine antibody, and/or other pharmacological or diagnosticcomponents, and/or written instructions explaining how to use the kit.59. A hybridoma that produces the monoclonal antibody or antigen bindingfragment thereof of claim
 47. 60. A method for making a hybridoma cellline that produces a multikine antibody or an antigen binding fragmentthereof that binds to two or more CC chemokines, comprising:sequentially immunizing a mammal with one CC chemokine, followed byboosting with one or more different CC chemokines, producing a hybridomacell line from said mammal, and isolating a hybridoma cell line thatproduces an antikine antibody.
 61. The method of claim 60 that producesa multikine antibody or an antigen binding fragment thereof that bindsto three or more CC chemokines that comprises boosting with two or moredifferent CC chemokines.
 62. A method for treating a disease, disorderor condition mediated by one or more CC chemokines comprisingadministering to a subject in need thereof the antikine antibody ofclaim 47 or an antigen binding fragment thereof.
 63. The method of claim62, wherein said disease, disorder, or condition is an inflammatorydisease or one mediated or associated with inflammation.
 64. The methodof claim 62, wherein said disease, disorder, or condition is anautoimmune disease.